BR122020001644B1 - COMPOSITION COMPRISING RECOMBINANT FOLLICLE STIMULATING HORMONE AND USE THEREOF FOR THE TREATMENT OF INFERTILITY - Google Patents

Abstract

The present invention relates to preparations including FSH, for example recombinant FSH, for use in the treatment of infertility.

Description

Divided from BR112014002884-2, deposited on 08.08.2012

[001] The present invention relates to pharmaceutical compositions and products for the treatment of infertility.

[002] Assisted reproductive technology (ART) techniques such as in vitro fertilization (IVF) are well known. These ART techniques generally require a controlled ovarian stimulation (COS) step, in which a group of follicles is stimulated to complete maturity. Standard COS regimens include the administration of gonadotropins such as follicle stimulating hormone (FSH) alone or in combination with luteinizing hormone (LH) activity to stimulate follicular development, typically with the administration of a GnRH analogue prior to and/or during stimulation to prevent premature LH surge. Pharmaceutical compositions commonly used for COS include recombinant follicle stimulating hormone (rFSH), urine-derived HSF, recombinant FSH + LH preparations, urine-derived menotropin [human menopausal gonadotropin (hMG)] and menopausal gonadotropin. highly purified human (HP-hMG). IVF may be associated with the risk of ovarian hyperstimulation syndrome (OHSS), which can be life-threatening in severe cases.

[003] The ability to predict a woman's potential response to controlled ovarian stimulation (COS) may allow for the development of individualized COS protocols. This may, for example, reduce the risk of OHSS in women predicted to have an excessive response to stimulation, and/or improve pregnancy responses in women classified as having poor responses. The serum concentration of anti-Müllerian hormone (AMH) is now established as a reliable marker of ovarian reserve. Reduced levels of AMH are correlated with reduced ovarian response to gonadotropins during COS. Additionally, elevated AMH levels are a good predictor of excessive ovarian response, and an indicator of risk for OHSS.

[004] In a preliminary study of women under 35 years of age undergoing ART, the CONSORT dosing algorithm (incorporating baseline FSH, BMI, age, and AFC) was used to predict the optimal starting dose of FSH for COS in women at risk of developing OHSS (Olivennes et al., 2009). Dose individualization led to adequate oocyte yield and good pregnancy rate. However, there were high cancellation coefficients in the low-dose group (75 IU FSH) due to inadequate response, and OHSS occurred in a significant proportion of patient doses.

[005] There is therefore a need for a composition for use in individualized COS protocols that provide adequate response to stimulation, and/or reduced risk of OHSS.

[006] As indicated above, standard COS protocols may include FSH administration. FSH is naturally secreted by the anterior pituitary gland and functions to support follicular development and ovulation. FSH comprises a 92 amino acid alpha subunit, also common to the other glycoprotein hormones LH and CG, and a unique 111 amino acid beta subunit of FSH that confers the biological specificity of the hormone (Pierce and Parsons, 1981). . Each subunit is post-translationally modified by the addition of complex carbohydrate residues. Both subunits carry 2 campor for n-linked glycan attachment, the alpha subunit at amino acids 52 and 78 and the beta subunit at amino acid residues 7 and 24 (Rathnam and Saxena, 1975, Saxena and Rathnam, 1976). FSH is thus glycosylated to about 30% by mass (Dias and Van Roey. 2001. Fox et al. 2001).

[007] FSH purified from post-menopausal human urine has been used for many years in the treatment of infertility; not only to promote ovulation in natural reproduction but also to provide oocytes for assisted reproductive technologies. Recombinant FSH (rFSH) products currently approved for ovarian stimulation, such as follitropin alpha (GONAL-F, Merck Serono / EMD Serono) and follitropin beta (PUREGON / FOLLISTIM, MSD / Schering-Plough), are derived from Chinese Hamster Ovary (CHO) cell. Currently, no rFSH product from a human cell line is commercially available.

[008] There is considerable heterogeneity associated with FSH preparations as it pertains to differences in the amounts of the various isoforms present. Individual FSH isoforms exhibit identical amino acid sequences but differ in the extent that they are post-translationally modified; Particular isoforms are characterized by heterogeneity of carbohydrate branch structures and different amounts of sialic acid (a terminal sugar) incorporation, both of which appear to influence the bioactivity of the specific isoform.

[009] The glycosylation of natural FSH is highly complex. The naturally derived glycans in pituitary FSH can contain a wide range of structures that can include combinations of mono-, bi-, tri-, and tetra-branched glycans (Pierce and Parsons, 1981. Ryan et al., 1987. Baenziger). and Green, 1988). Glycans can carry additional modifications: core fucosylation, glucosamine bisection, extended chains with acetyl lactosamine, partial or complete sialylation, sialylation with α2,3 and α2,6 bonds, and sulfated galactosamine substituted for galactose (Dalpathado et al. , 2006). Additionally, there are differences between the distributions of glycan structures in individual glycosylation fields. A comparable level of glycan complexity has been observed in FSH derivatives from the serum of subjects and from the urine of postmenopausal women (Wide et al., 2007).

[0010] The glycosylation of recombinant FSH products reflects the range of glycosyl transferases present in the host cell lineage. Commercially offered rFSH products are derived from engineered Chinese Hamster Ovarian Cells (CHO Cells). The range of glycan modifications in CHO cell-derived rFSH is more limited than those found in natural products. Examples of the reduced glycan heterogeneity found in CHO cell-derived rFSH include the lack of Glucosamine bisection and a reduced content of core fucosylation and acetyl lactosamine extensions (Hard et al., 1990). In addition, CHO cells are only able to add sialic acid using the α2,3 linkage (Kagawa et al, 1988, Takeuchi et al, 1988, Svensson et al., 1990); CHO cell-derived rFSH only includes α2,3-linked sialic acid and does not include α2,6-linked sialic acid.

[0011] Thus CHO cell-derived FSH is different from naturally produced FSH (e.g. human pituitary/serum/urinary FSH) which contains glycans with a mixture of sialic acid bound to α2,3 and α2, 6, with the predominance of the trainer.

[0012] Additionally, it has also been shown that the commercially offered recombinant FSH preparation differs in the amounts of FSH with an isoelectric point (pI) of below 4 (considered the acidic isoforms) when compared to FSH from pituitary, serum or urine. postmenopausal (Ulloa-Aguirre et al. 1995). The amount of acidic isoforms in the urinary preparations was much higher compared to the CHO cell-derived recombinant products Gonal-f (Merck Serono) and Puregon (Schering Plow) (Andersen et al. 2004). This should reflect a low molar content of sialic acid in recombinant FSH since the content of sulfate-modified negatively charged glycan is low in recombinant FSH. The lower sialic acid content compared to natural FSH is a feature of both commercially offered recombinant FSH products and may reflect a limitation in the manufacturing process.

[0013] Life in the circulation of FSH has been documented for materials from a variety of sources. Some of said materials have been fractionated on the basis of overall molecular charge, as characterized by their pI, where more acid equates to a higher negative charge. As previously determined the largest contributor to overall molecular charge is the total sialic content of each FSH molecule. For example, rFSH (Organon) has a sialic acid content of around 8 mol/mol, whereas urine-derived FSH has a higher sialic acid content (de Leeuw et al. 1996). The corresponding plasma clearance coefficients in the rat are 0.34 and 0.14 mL/min (Ulloa-Aguirre et al. 2003). In another example where a recombinant FSH sample was fractionated into high and low pl fractions, the in vivo potency of the high pl fraction (lower sialic acid content) was reduced and it had a shorter plasma half-life (D 'Antonio et al. 1999). It has also been reported that the more basic FSH circulating during the later stages of the ovulation cycle is due to the downregulation of α2,3 sialyl-transferase in the anterior pituitary that is caused by higher levels of estradiol (Damian-Matsumara et al. 1999. Ulloa-Aguirre et al. 2001). Results for α2,6 sialyl transferase were not reported.

[0014] Thus, as determined above, recombinant proteins expressed using the CHO system will differ from their natural counterparts in their types of terminal sialic acid bonds. This is an important consideration in the production of biological products for pharmaceutical use as the carbohydrate fractions can contribute to the pharmacological attributes of the molecule.

[0015] The present applicants have developed a human-derived recombinant FSH which is the subject of International Patent Application No. PCT/GB2009/000978, published as WO2009/127826A. Recombinant FSH with a mixture of both sialic acid bound to α2,3 and α2,6 was produced by genetically engineering the human cell line to express not only rFSH but also α2,3 sialyltransferase. The expressed product is highly acidic and carries a mixture of both sialic acids bound to α2,3- and α2,6; the latter provided by endogenous sialyl transferase activity. It has been observed that the type of sialic acid binding, α2,3- or α2,6-, can have a dramatic influence on the biological clearance of FSH. Recombinant FSH with a mixture of both sialic acid bound to α2,3 and α2,6 has two advantages over rFSH expressed in conventional CHO cells: first, the material is more highly sialylated by virtue of the combined activities of the two sialyltransferases; and second material most closely resembles natural FSH. This should be more biologically appropriate compared to a CHO cell-derived recombinant products that produced only α2,3-linked sialic acid (Kagawa et al, 1988, Takeuchi et al, 1988, Svensson et al., 1990) and have reduced acid content. sialic (Ulloa-Aguirre et al. 1995., Andersen et al. 2004).

[0016] The rFSH product described in International Patent Application No. PCT/GB2009/000978 contains branched glycan fractions. FSH comprises glycans (attached to FSH glycoproteins) and said glycans can contain a wide variety of structures. As is well known in the art, branching (of a glycan) can occur with the result that the glycan can have 1, 2, 3, 4 or more terminal sugar residues or "branching"; glycans with 1, 2, 3 or 4 terminal sugar residues or "branching" are referred to as mono-branched, di-branched, tri-branched or tetra-branched structures, respectively. Glycans may have the presence of sialylation in the mono-branched and/or di-branched and/or tri-branched and/or tetra-branched structures. An example of rFSH described in International Patent Application No. PCT/GB2009/000978 includes monosialylated, disialylated, trisialylated and tetrasialylated glycan structures with relative amounts as follows: 9-15% monosialylated; 27 - 30% di--sialylated; 30 - 36% tri-sialylated and 25 - 29% tetra-sialylated. As is well known, a monosialylated glycan structure carries a sialic acid residue; a di-sialylated glycan backbone carries two sialic acid residues; a trisialylated glycan backbone carries three sialic acid residues; and a tetrasialylated glycan backbone carries four sialic acid residues. Here, terminology such as "X% mono-sialylated", "X% di-sialylated", "X% tri-sialylated" or "X% tetrasialylated" refers to the number of glycan structures in FSH that are mono-, di, tri or tetra sialylated (respectively), expressed as a percentage (X%) of the total number of glycan structures in FSH that are sialylated in any mode (ports sialic acid). Thus, the phrase "30 - 36% tri-sialylated glycan structures" means that, of the total number of glycan structures in FSH that carry sialic acid residues (i.e. are sialylated), 30 to 36% of said glycan structures are trisialylated (carrying three sialic acid residues). Applicants have surprisingly observed that FSH having a specific amount of tetrasialylated glycan structures (which is different from the example of the rFSH product described in PCT/GB2009/000978 mentioned above) is markedly more potent than the recombinant FSH products which are currently on the market. The amino acid sequence of applicants' products is the native sequence and is identical to existing natural human FSH and CHO-derived rFSH products. However, the present inventors have observed that human-derived recombinant FSH products (i.e., recombinant FSH produced or expressed in a human cell line e.g., produced by genetic engineering of the human cell line) that have a mixture of both sialic acid linked to α2,3 and α2,6 and/or the specific amount of tetrasialylated glycan structures can be particularly effective when used in COS protocols (e.g. individualized).

[0017] In accordance with the present invention in a first aspect there is provided a product (e.g. a pharmaceutical composition) comprising follicle stimulating hormone (FSH) for use in treating infertility in a patient (e.g. a patient having a of serum AMH of 0.05 pmol/L or above, e.g. 0.5 pmol/L or above), wherein the product comprises a dose of, or a dose equivalent to, 1-24 μg, e.g. 2-24 µg, e.g. 2 to 15 µg, of human-derived recombinant FSH. Preferably the product comprises a dose of, or a dose equivalent to, 4.5 to 12.5 µg, for example 5 to 12.5 µg, for example 6 to 12.5 µg, for example 6.3 to 12.5 µg 10.5 µg, human-derived recombinant FSH.

[0018] In accordance with the present invention there is provided a product (e.g. the pharmaceutical composition) comprising follicle stimulating hormone (FSH) for use in treating infertility in a patient having a serum AMH level of <15 pmol/L (e.g. 0.05 pmol/L to 14.9 pmol/L), wherein the product comprises a dose (e.g. daily), or dose equivalent to, 9 to 14 μg, e.g. 11 to 13 μg , for example, 12 μg of human-derived recombinant FSH. Preferably the FSH is a recombinant FSH ("rFSH" or "recFSH"). Preferably the FSH is a human-derived recombinant FSH cell line. The dose provides an effective response while minimizing the risk of OHSS. Preferably the infertility treatment comprising a step of determining (e.g. measuring) the patient's serum AMH level, and administering the dose to a patient having a serum AMH level of <15 pmol/L (e.g. 0 .05 pmol/L to 14.9 pmol/L).

[0019] In accordance with the present invention in a further aspect there is provided a product (e.g. a pharmaceutical composition) comprising follicle stimulating hormone (FSH) for use in treating infertility in a patient having a serum AMH level of >15 pmol/L, wherein the product comprises a dose of (e.g., daily), or dose equivalent to, 5 to 12.5 µg, e.g. 6 to 10.5 µg of human-derived recombinant FSH. Preferably the FSH is a recombinant FSH ("rFSH" or "recFSH"). Preferably the FSH is a human-derived recombinant FSH cell line. The dose provides an effective response while minimizing the risk of OHSS. Preferably the treatment of infertility comprising a step of determining (e.g. measuring) the patient's serum AMH level, and administering the dose to a patient having a serum AMH level of >15 pmol/L. In one embodiment, the product is for use in treating infertility in a patient having a serum AMH level of 15 to 24.9 pmol/L, and the product is for administration at a dose of (e.g., daily), or dose equivalent to, 5 to 12 µg, e.g. 7 to 12 µg, e.g. 8.7 to 10 µg, of human-derived recombinant FSH (preferably 9 to 10 µg of human-derived recombinant FSH). In the present embodiment, the treatment of infertility may comprise a step of determining (e.g., measuring) the patient's serum AMH level, and administering the dose to a patient having a serum AMH level of 15 to 24.9 pmol. /L. In another embodiment, the product is for use in treating infertility in a patient having a serum AMH level of 25 to 34.9 pmol/L, and the product is for administration at a dose of (e.g., daily), or dose equivalent to, 5 to 12 µg, e.g. 6 to 9 µg, e.g. 7 to 8 µg human-derived recombinant FSH (preferably 7.3 to 8 µg human-derived recombinant FSH). In the present embodiment, the treatment of infertility may comprise a step of determining (e.g., measuring) the patient's serum AMH level, and administering the dose to a patient having a serum AMH level of 25 to 34.9 pmol. /L. In another embodiment, the product is for use in treating infertility in a patient having a serum AMH level of > 35 pmol/L, and the product is for administration at a dose of (e.g., daily), or equivalent dose a, 5 to 11 µg, e.g. 6.3 to 7 µg, of human-derived recombinant FSH (preferably 6 to 7 µg of human-derived recombinant FSH). In the present embodiment, infertility treatment may comprise a step of determining (e.g., measuring) the patient's serum AMH level, and administering the dose to a patient having a serum AMH level of > 35 pmol/L.

[0020] The above doses may be for the treatment of infertility in the patient's (individual) first stimulation protocol. It will be noted that for additional stimulation cycles, doses may be adjusted according to the current ovarian response in the first cycle.

[0021] Applicants have noted that it is generally necessary to recover in the nine oocyte region in order to allow for the selection of two high quality oocytes for transfer.

[0022] Applicants noted that for subjects having low AMH (AMH < 15 pmol/L per litre) a relatively high dose of recombinant FSH is required (eg, 12 μg) to achieve this. At this dose, 8 to 14 oocytes will be recovered from 60% of individuals with low AMH. This is an unexpected and significant improvement in the treatment of low AMH subjects treated with 150 IU Gonal-f, where 8 to 14 oocytes are recovered from only 33% of subjects. Applicants have observed that there is no need to adjust said dose according to the patient's body weight.

[0023] However, 60% of the population (and 80% of women under 30 treated for infertility) have high AMH (ie, AMH of >15 pmol/L). For such individuals it is generally relatively certain to recover an average of 9 to 11 oocytes; the problem with pacing protocols is the risk of OHSS. Applicants have observed that in patients dosed with low doses of recombinant human FSH there is a relationship between retrieved oocytes and subject body weight. This means that there may be a risk associated with treatment with a fixed dose of FSH (which is usual in the art). The present applicants have established a relationship between FSH dose and AMH level and subject weight that provides an improved safety profile (reduced risk of OHSS) with acceptable or improved oocyte retrieval compared to known treatment protocols (see example 10). ).

[0024] In accordance with the present invention in a further aspect there is provided a product (e.g. a pharmaceutical composition) comprising follicle stimulating hormone (FSH) for use in treating infertility in a patient having a serum AMH level of > 15 pmol/L, where the product is for administration at a dose of (eg, daily), or a dose equivalent to, 0.09 to 0.19 μg (eg, 0.09 to 0.17 μg) of Human-derived recombinant FSH per kg of patient body weight. Preferably the infertility treatment comprises a step of determining (e.g. measuring) the patient's serum AMH level, and administering the dose to a patient having a serum AMH level of >15 pmol/L. In one embodiment, the product is for use in treating infertility in a patient having a serum AMH level of 15 to 24.9 pmol/L, and the product is for administration at a dose of (e.g., daily), or dose equivalent to, 0.14 to 0.19 µg human-derived recombinant FSH (preferably 0.15 to 0.16 µg human-derived recombinant FSH) per kg of patient body weight. In the present embodiment, the treatment of infertility may comprise a step of determining (e.g., measuring) the patient's serum AMH level, and administering the dose to a patient having a serum AMH level of 15 to 24.9 pmol. /L. In another embodiment, the product is for use in treating infertility in a patient having a serum AMH level of 25 to 34.9 pmol/L, and the product is for administration at a dose of (e.g., daily), or dose equivalent to, 0.11 to 0.14 µg human-derived recombinant FSH (preferably 0.12 to 0.13 µg human-derived recombinant FSH) per kg of patient body weight. In the present embodiment, the treatment of infertility may comprise a step of determining (e.g., measuring) the patient's serum AMH level, and administering the dose to a patient having a serum AMH level of 25 to 34.9 pmol. /L. In yet a further embodiment, the product is for use in treating infertility in a patient having a serum AMH level of >35 pmol/L, and the product is for administration at a dose of (e.g., daily), or dose equivalent to, 0.10 to 0.11 μg of human-derived recombinant FSH per kg of patient body weight. In the present embodiment, infertility treatment may comprise a step of determining (e.g., measuring) the patient's serum AMH level, and administering the dose to a patient having a serum AMH level of > 35 pmol/L. Preferably the FSH is a recombinant FSH ("rFSH" or "recFSH"). Preferably the FSH is a human-derived recombinant FSH cell line. The doses provide an effective response while minimizing the risk of OHSS.

[0025] The above doses may be for the treatment of infertility in the patient's (individual) first stimulation protocol. It will be noted that for additional stimulation cycles, doses may be adjusted according to the current ovarian response in the first cycle.

[0026] In accordance with the present invention in a further aspect there is provided a product (e.g. a pharmaceutical composition) comprising follicle stimulating hormone (FSH) for use in treating infertility in a patient having a serum AMH level of <15 pmol/L, where the product is for administration at a dose of (e.g., daily), or dose equivalent to, 0.15 to 0.21 μg, (e.g., 0.19 to 0.21 μg ) of human-derived recombinant FSH per kg of patient body weight. Preferably the infertility treatment comprises a step of determining (e.g. measuring) the patient's serum AMH level, and administering the dose to a patient having a serum AMH level of <15 pmol/L.

[0027] However, it is not necessary for patients having a serum AMH level of <15 pmol/L to be dosed by body weight. It will be appreciated that said doses can be readily converted to treat patients dosed according to their BMI, using conversions well known in the art.

[0028] The product (e.g. pharmaceutical composition) may be for use in treating infertility in a patient having serum AMH of 5.0 - 14.9 pmol/L, wherein the product comprises a dose of, or dose equivalent to, 6 to 18 µg, e.g. 8 to 11 µg, e.g. 8.5 to 10.2 µg of human-derived recombinant FSH. The product may be for use in treating infertility in a patient having serum AMH 15.0 - 29.9 pmol/L, wherein the product comprises a dose of, or a dose equivalent to, 4.8 to 15 µg, e.g. 6 to 9 µg, e.g. 6.8 to 8.5 µg of human-derived recombinant FSH. The product may be for use in treating infertility in a patient having serum AMH 30 - 44.9 pmol/L, wherein the product comprises a dose of, or a dose equivalent to, 3.6 to 12 μg, for example , 4 to 7 µg, e.g. 5.1 to 6.8 µg of human-derived recombinant FSH. The product may be for use in treating infertility in a patient having serum AMH 45 pmol/L or more, wherein the product comprises a dose of, or a dose equivalent to, 2 to 9 μg, e.g. 2.4 to 9 µg (eg, 3.4 to 5.1 µg) or 2 to 5 µg of human-derived recombinant FSH. The product may comprise follicle stimulating hormone (FSH) for use in treating infertility in a patient having serum AMH of 5 pmol/L or less, wherein the product comprises a dose of, or a dose equivalent to, 7.2 to 24 µg, e.g. 10 to 15 µg, e.g. 10.2 to 13.6 µg, of human-derived recombinant FSH. The product may be for use in treating infertility in a patient where the product comprises a dose of, or dose equivalent to, 4.8 to 18 μg, e.g. 6 to 11 μg, e.g. 6.8 to 10 .2 µg human-derived recombinant FSH. Preferably the FSH is a recombinant FSH ("rFSH" or "recFSH"). Preferably the FSH is a human-derived recombinant FSH cell line.

[0029] Preferably the rFSH (e.g., human-derived recombinant FSH cell line) includes α2,3- and α2,6 sialylation. The FSH (rFSH) for use in accordance with the present invention may have 1% to 99% of the total sialylation being α2,3-sialylation. The FSH (rFSH) according to the present invention may have 1% to 99% of the total sialylation being α2,6-sialylation. Preferably, 50 to 70%, for example 60 to 69%, for example about 65%, of the total sialylation is α2,3-sialylation. Preferably 25 to 50%, for example 30 to 50%, for example 31 to 38%, for example about 35%, of the total sialylation is 2,6 sialylation.

[0030] Preferably the rFSH (e.g. human-derived recombinant FSH cell line) includes mono-, di-, tri- and tetra-sialylated glycan structures, wherein 15-24%, e.g. 17-23% of the sialylated glycan structures are tetrasialylated glycan structures (e.g., as shown by WAX analysis of charged glycans, as determined in the Examples below). FSH comprises glycans (attached to FSH glycoproteins). It is well known that the glycans in FSH can contain a wide variety of structures. Said may include combinations of mono-, bi-, tri- and tetra-branched glycans. Here, terminology such as "X% of sialylated glycan structures are tetrasialylated glycan structures" refers to the number of glycan structures in FSH that are tetrasialylated, that is, carry four sialic acid residues, expressed as the percentage (X%) of the total number of glycan structures in FSH that are sialylated in any mode (carry sialic acid). Thus, the phrase "15-24% of sialylated glycan structures are tetrasialylated glycan structures" means that of the total number of glycan structures in FSH that carry sialic acid residues (i.e. are sialylated), 15 to 24 % of said glycan structures are tetra sialylated (they carry four sialic acid residues).

[0031] rFSH can be present as a single isoform or as a mixture of isoforms.

[0032] Applicants have noted that "individualized" COS protocols in which specific doses of recombinant FSH having specific characteristics are used to treat patients based on their specific AMH levels, thereby increasing the likelihood of adequate response to stimulation (e.g., in patients having a low potential response), and/or reduced risk of OHSS (eg, in patients classified as high or overresponsive).

[0033] The serum AMH level can be determined (e.g., measured) by any method known in the art. Preferably the serum AMH level is measured using the AMH Gen-II enzyme linked immunosorbent assay, a kit (Beckman Coulter, Inc., Webster, Texas). This test can detect AMH concentrations of greater than 0.57 pmol/L with a lower limit of quantitation of 1.1 pmol/L. Other tests can be used.

[0034] Here, serum AMH values are generally quoted in terms of pmol/L. This can be converted to ng/mL using the conversion equation 1ng/mL AMH = 7.1 pmol/L AMH.

[0035] Here the terms "patient" and "individual" are used interchangeably.

[0036] The product (e.g. pharmaceutical composition) preferably comprises a daily dose of, or a daily dose equivalent to, the amounts of human rFSH derivatives defined above, herein, and in the claims. The (daily) dose may be an initial dose (ie, it may be reduced, increased, or maintained during treatment).

[0037] The product (e.g. pharmaceutical composition) may be for the (daily) administration of FSH starting on day one of treatment and continuing for seven to thirteen days, e.g. nine to thirteen days, e.g. 10 to 13 days, for example 10 to 11 days. The product (e.g. pharmaceutical composition) may be for administration 12 to 16, e.g. 13 to 15, e.g. 14 days after administration of (e.g. after initiation of administration of, e.g. after initiation of of daily administration) of a GnRH agonist (e.g. Synarel, Lupron, Decapeptil). The product (e.g. pharmaceutical composition) may be for administration with a GnRH agonist. The product (e.g. pharmaceutical composition) may be for administration prior to administration of a GnRH antagonist (e.g. ganirelix, cetrorelix), for example for administration five or six days prior to administration of a GnRH antagonist. The product (e.g. pharmaceutical composition) may be for administration with a GnRH antagonist. Preferably the product (e.g. pharmaceutical composition) is for administration prior to administration of a high (ovulatory) dose of hCG (e.g. 4,000 to 11,000 IU hCG, e.g. 5,000 IU hCG, 10,000 IU hCG etc.; or 150 to 350 micrograms of recombinant hCG, e.g. 250 micrograms of recombinant hCG) to induce final follicular maturation.

[0038] It will be noted that the product may be for dosing at frequencies more (or less) than daily, in which case the relevant doses will be equivalent to the (daily) doses specified here.

[0039] Here the term "infertility treatment" includes infertility treatment by controlled ovarian stimulation (COS) or methods that include a stage or stage of controlled ovarian stimulation (COS), e.g. Intrauterine Insemination (IUI), fertilization in vitro (IVF), or intracytoplasmic sperm injection (ICSI). The term "infertility treatment" includes treatment of infertility by ovulation induction (OI) or by methods that include an ovulation induction (OI) stage or stage. The term "infertility treatment" includes treatment of infertility in a subject having tubal or unexplained infertility, including treatment of infertility in a subject having endometriosis, e.g., stage I or stage II endometriosis, and/or in a subject having anovulatory infertility. , for example, WHO type II anovulatory infertility, and/or in an individual with a partner with male factor infertility. The product (or composition) may be for (use in) the treatment of infertility (and/or for controlled ovarian stimulation) in a subject having endometriosis, for example, in a subject having stage I or stage II endometriosis, as defined by the The American Society for Reproductive Medicine (ASRM) classification system for the various stages of endometriosis, (stage IV the most severe; stage I the least severe) [American Society for Reproductive Medicine. Revised American Society for Reproductive Medicine classification of endometriosis: 1996. Fertil Steril 1997; 67,817 821.].

[0040] The product (composition) may be for (use in) the treatment of infertility (and/or for controlled ovarian stimulation) in an individual having a normal serum FSH level of 1 to 16 IU/L, e.g. 1 to 12 IU/L, in the early follicular phase.

[0041] The product (composition) may be for (use in) the treatment of infertility (and/or for controlled ovarian stimulation) in an individual aged 18 to 42 years, eg 25 to 37 years. The product may be for (use in) the treatment of infertility (and/or for controlled ovarian stimulation) in an individual having BMI >1 and BMI < 35 kg/m2, for example, an individual having BMI >18 and BMI < 25 kg/m2, for example, an individual having a BMI >20 and a BMI < 25 kg/m2.

[0042] The rFSH may preferably include 27 - 33%, for example 30 - 32%, trisialylated glycan structures. The rFSH may preferably include 24 - 33%, for example 26 - 30%, disialylated glycan structures. The rFSH may preferably include 12 - 21%, for example 15 - 17%, monosialylated glycan structures. rFSH preferably includes monosialylated, disialylated, trisialylated and tetrasialylated glycan structures with relative amounts as follows: 15 to 17% monosialylated; 26 - 30% di--sialylated; 27 - 33% (e.g. 29 to 32%, e.g. 30-32%, e.g. 30 to 31%) tri-sialylated and 17 - 23% tetra-sialylated (e.g. as shown by WAX analysis of charged glycans, as determined in the Examples). The rFSH may include from 0 to 7%, for example 0.1 to 7%, for example 3 to 6%, for example 5 to 6%, neutral sialylated structures. FSH comprises glycans (attached to FSH glycoproteins). Here, terminology such as "X% mono-sialylated", "X% di-sialylated", "X% tri-sialylated" or "X% tetrasialylated" refers to the number of glycan structures in FSH which are mono-, di, tri or tetra sialylated (respectively), expressed as the percentage (X%) of the total number of glycan structures in FSH that are sialylated in any mode (carry sialic acid). Thus, the phrase "27 - 33% trisialylated glycan structures" means that, of the total number of glycan structures in FSH that carry sialic acid residues (i.e. are sialylated), 27 to 33% of said glycans are trisialylated (carry three sialic acid residues).

[0043] rFSH may have a sialic acid content [expressed in terms of a ratio of moles of sialic acid to moles of protein] of 6 mol/mol or more, for example between 6 mol/mol and 15 mol/mol e.g. between 8 mol/mol and 14 mol/mol, e.g. between 10 mol/mol and 14 mol/mol, e.g. between 11 mol/mol and 14 mol/mol, e.g. between 12 mol/ mol and 14 mol/mol, for example between 12 mol/mol and 13 mol/mol. rFSH can be produced or expressed in the human cell line.

[0044] The FSH (rFSH) for use in accordance with the present invention may have 1% to 99% of the total sialylation being α2,3-sialylation. rFSH may have 10% or more of the total sialylation being α2,3-sialylation. For example, 20, 30, 40, 50, 60, 70, 80 or 90% or more of the total sialylation can be α2,3-sialylation. The rFSH may preferably include α2,3-sialylation in an amount that is from 50 to 70% of the total sialylation, for example, from 60 to 69% of the total sialylation, for example, from 63 to 67% , for example around 65% of the total sialylation. FSH (rFSH) for use in accordance with the present invention may have 1% to 99% of the total sialylation being α2,6-sialylation. The rFSH (or rFSH preparation) of the present invention may have 5% or more, for example 5% to 99%, of the total sialylation being α2,6-sialylation. rFSH can have 50% or less of the total sialylation being α2,6-sialylation. The rFSH may preferably include α2,6-sialylation in an amount that is from 25 to 50% of the total sialylation, for example, from 30 to 50% of the total sialylation, for example, from 31 to 38% , for example around 35% of the total sialylation. By sialylation is meant the amount of sialic residues present in the carbohydrate structures of FSH. α2,3-sialylation means sialylation at position 2,3 (as is well known in the art) and α2,6 sialylation at position 2,6 (also well known in the art). Thus "% of the total sialylation can be α 2,3 sialylation" refers to the % of the total number of sialic acid residues present in FSH that are sialylated at the 2,3 position. The term "% of total sialylation being α2,6-sialylation" refers to the % of the total number of sialic acid residues present in FSH that are sialylated at the 2,6 position.

[0045] rFSH may have a sialic acid content (amount of sialylation per FSH molecule) of (based on protein mass rather than protein mass plus carbohydrate) of 6% or more (e.g., between 6% and 15%, for example between 7% and 13%, for example between 8% and 12%, for example between 11% and 15%, for example between 12% and 14%) by mass.

[0046] The rFSH may be rFSH or an rFSH preparation in which 16% or less (e.g., 0.1 to 16%) of the glycans comprise (e.g., carry) bisection of N-acetylglucosamine (bisection of GlcNAc or of bisGlcNAc). Preferably the rFSH (or rFSH preparation) is an rFSH or rFSH preparation in which 8 to 14.5% of the glycans comprise (e.g. carry) N-acetylglucosamine bisection (GlcNAc or bisGlcNAc bisection).

[0047] It will be understood that FSH comprises glycans attached to FSH glycoproteins. It will also be understood that 100% glycans refers to or means all glycans attached to FSH glycoproteins. Thus, here, the terminology "8 to 14.5% of the glycans comprise (carry) bisection of N-acetylglucosamine" means that 8 to 14.5% of the total number of glycans attached to FSH glycoproteins include/carry N-bisection -acetylglucosamine; "16% or less of the glycans comprise (carry) N-acetylglucosamine bisection" means that 16% or less of the total number of glycans attached to FSH glycoproteins include/carry N-acetylglucosamine bisection, and so on.

[0048] Applicants have observed that recombinant FSH (rFSH preparation; rFSH compositions) in which 16% or less (e.g., 8 to 14.5%) of the glycans not comprising FSH glycoproteins bearing GlcNac bisection may have pharmacological properties. - advantageous cokinetics. It is believed that the advantageous properties may arise because the amount of glycans carrying GlcNac bisection is similar to that in Bravelle human urine-derived products, which is relatively lower than that of other recombinant FSH preparations such as those described. in WO2012/017058.

[0049] The rFSH (or rFSH preparation) may be an rFSH or rFSH preparation in which 20% or more of the glycans comprise (e.g., carry) N-Acetylgalactosamine (GalNAc), for example, in which 20% or more of the glycans comprise (e.g. carry) a terminal GalNAc. Preferably the rFSH (or rFSH preparation) is an FSH or FSH preparation in which 40 to 55%, e.g. 42% to 52%, of the glycans comprise (e.g. carry) GalNAc. Preferably the rFSH (or rFSH preparation) is an FSH or FSH preparation in which 40 to 55%, e.g. 42% to 52%, of the glycans comprise (e.g., carry) terminal GalNAc.

[0050] It will be understood that FSH comprises glycans attached to FSH glycoproteins. It will also be understood that 100% glycans refers to or means all glycans attached to FSH glycoproteins. Thus, here, the terminology "wherein 20% or more of the glycans comprise (e.g., carry) GalNAc" means that 20% or more of the total number of glycans attached to FSH glycoproteins include/carry N-Acetylgalactosamine ( GalNAc); "40 to 55%, e.g. 42% to 52%, of the glycans comprise (e.g. carry) terminal GalNAc" means that 40 to 55%, e.g. 42% to 52%, of the total number of fixed glycans FSH glycoproteins include/carry terminal GalNAc, and so on.

[0051] It appears that the availability of α2,6 binding increases the number of tetra sialylated structures compared to CHO cell-derived products that have only α2,3 binding available. Applicants also noted that their rFSH is distinct from other approved products by virtue of the sugar composition: it includes, or may include, a specific amount of GalNac. This may be linked to tetrasialylation and potency due to the fact that 2,6 sialylation is associated with GalNac. In other words, the present inventors have developed an rFSH product that includes specific features (2,6 binding fields, GalNac) that provide rFSH with a high degree of sialylation, which appears to lead to enhanced potency in vivo.

[0052] The rFSH (or rFSH preparation) may have 16 to 24% of the glycans comprising (e.g. terminal) 1 fucose-lewis, e.g. 16.5 to 18% of the glycans comprising (e.g. terminal) 1 fucose-lewis. The rFSH (or rFSH preparation) may have 1.5 to 4.5%, e.g. 2 to 4%, e.g. 3.7%, of the glycans comprising (e.g. terminal) 2 fucose -lewis .

[0053] The fucose-lewis content may have an effect on potency.

[0054] rFSH can be produced or expressed in a human cell line, for example a Per.C6 cell line, a HEK293 cell line, an HT1080 cell line etc. This can simplify (and make more efficient) the method of production by virtue of the manipulation and control of, for example, the cell growth medium to retain sialylation may be less fundamental than known processes. The method may also be made more efficient by the fact that there is little basic rFSH produced compared to the production of known rFSH products; more acidic rFSH is produced and separation/removal of basic FSH is less problematic. rFSH can be produced or expressed in a PER.C6® cell line, a PER.C6® derived cell line, or a PER.C6® modified cell line. rFSH that is produced or expressed in a human cell line (e.g. PER.C6® cell line, HEK293 cell line, HT1080 cell line etc.) will include some α2,6-linked sialic acid (α2,6 sialylation) provided by endogenous sialyl transferase activity [from the cell line] and will include some α2,3-linked sialic acids (α2,3 sialylation) provided by endogenous sialyl transferase activity. The cell line can be modified using α2,3-sialyltransferase. The cell line can be modified using α2,6-sialyltransferase. Alternatively or additionally, rFSH may include α2,6-linked sialic acids (α2,6 sialylation) provided by endogenous sialyl transferase activity [from the cell line]. Here, the term "human-derived recombinant FSH" means recombinant FSH that is produced or expressed in a human cell line (e.g., recombinant FSH produced by genetic engineering of the human cell line).

[0055] rFSH can be produced using α2,3- and/or α2,6-sialyltransferase. In one example, rFSH is produced using α2,3-sialyltransferase. rFSH may include α2,6-linked sialic acids (α2,6 sialylation) provided by endogenous sialyl transferase activity.

[0056] The product may be a pharmaceutical composition. The pharmaceutical composition is for the treatment of infertility. Infertility treatment may comprise assisted reproductive technologies (ART), ovulation induction or intrauterine insemination (IUI). The pharmaceutical composition can be used, for example, in medical indications where known FSH preparations are used.

[0057] The product or composition may be formulated into compositions well known for any route of drug administration, e.g., oral, rectal, parenteral, transdermal (e.g. patch technology), intravenous, intramuscular, subcutaneous, intrasternal. , intravaginally, intraperitoneally, locally (powders, ointments or drops) or as a buccal or nasal spray. A typical composition comprises a pharmaceutically acceptable carrier, such as aqueous solution, non-toxic excipients, including salts and preservatives, buffers and the like, as described in Remington's Pharmaceutical Sciences fifteenth edition (Matt Publishing Company, 1975), at pages 1405 to 1412 and 1461 - 87, and in the fourteenth edition of the national formulary XIV (American Pharmaceutical Association, 1975), among others.

[0058] Examples of suitable aqueous and non-aqueous pharmaceutical carriers, diluents, solvents, or pharmaceutical vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), carboxymethyl cellulose and suitable mixtures thereof, vegetable oils (such as such as olive oil), and injectable organic esters such as ethyl oleate.

[0059] The compositions of the present invention may also contain additives such as but not limited to preservatives, wetting agents, emulsifying agents, surfactants and dispersing agents. Antibacterial and antifungal agents can be included to prevent the growth of microbes and include, for example, m-cresol, benzyl alcohol, paraben, chlorobutanol, phenol, sorbic acid, and the like. If a preservative is included, benzyl alcohol, phenol and/or m-cresol are preferred; however, the preservative is by no means limited to said examples. Additionally, it may be desirable to include isotonic agents such as sugars, sodium chloride, and the like. The product or composition may further comprise a salt comprising a pharmaceutically acceptable alkali metal cation selected from the group consisting of Na+- or K+- salts, or combinations thereof. Preferably the salt is a Na+- salt, for example NaCl or Na2SO4.

[0060] Preferably the product or composition comprises recombinant FSH and one or more of Polysorbate 20, L-methionine, phenol, disodium sulfate and sodium phosphate buffer.

[0061] In some cases, to effect prolonged action it is desirable to reduce the absorption of FSH (and other active ingredients, if present) from subcutaneous or intramuscular injection. This can be accomplished by using a liquid suspension of crystalline or amorphous material with poor water solubility. The absorption coefficient of FSH then depends on its dissolution coefficient which, in turn, may depend on crystal size and crystal form. Alternatively, delayed absorption of a parenterally administered form of FSH is accomplished by dissolving or suspending the FSH combination in an oil vehicle. Injectable depot forms can be produced by forming microcapsule matrices of FSH (and other agents, if present) in biodegradable polymers such as polylactide-polyglycolide. Depending on the FSH to polymer ratio and the nature of the particular polymer employed, the FSH release coefficient can be controlled. Examples of other biodegradable polymers include polyvinylpyrrolidone, poly(orthoesters), poly(anhydrides) and the like. Depot injectable formulations are also prepared by entrapping FSH in liposomes or microemulsions that are compatible with body tissues.

[0062] Injectable formulations can be sterilized, for example, by filtering through a bacterial retention filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable media shortly before of use. Injectable formulations may be supplied in any suitable container, for example, vial, pre-filled syringe, injection cartridges, and the like.

[0063] The product or composition can be formulated for single use or for multiple use (multiple dose). If the product or composition is formulated for multiple use, it is preferred that a preservative be included. If a preservative is included, benzyl alcohol, phenol and/or m-cresol are preferred; however, the preservative is by no means limited to said examples. The product or composition formulated for single use or for multiple use may further comprise a salt comprising a pharmaceutically acceptable alkali metal cation selected from the group consisting of salts of Na+- or K+-, or a combination thereof. Preferably the salt is a Na+- salt, for example NaCl or Na2SO4.

[0064] The product or composition may be included in a container such as a vial, pre-filled cartridge (e.g. for single administration or multiple use) or an injection device such as a "pen", e.g. for administration of multiple doses.

[0065] The product or composition may be a formulation (eg injectable formulation) including FSH (optionally with hCG, LH, LH etc activity). LH activity, if present, may originate from LH or from human chorionic gonadotropin, hCG. If there is more than one active ingredient (i.e. FSH and, for example, hCG or LH) these may be suitable for administration separately or together. If administered separately, administration may be sequential. The product can be supplied in any suitable packaging. For example, a product may include a number of containers (eg, pre-filled syringes or vials) containing either FSH or hCG, or a combination (or combination) of both FSH and hCG. The hCG can be either recombinant hCG or urinary hCG. If the product includes a number of containers (eg pre-filled syringes or vials) containing FSH, for example recombinant FSH, each container may include the same amount of FSH. One or more containers may include different amounts of FSH. Syringes or vials may be packaged in a blister pack or other means to maintain sterility. Any product may optionally contain instructions for using the FSH formulations (and eg hCG if present). The pH and exact concentration of the various components of the pharmaceutical composition are adjusted according to routine practice in this field. See GOODMAN and GILMAN's THE PHARMACOLOGICAL BASIS FOR THERAPEUTICES, 7th ed. In a preferred embodiment, the compositions of the present invention are provided as compositions for parenteral administration. General methods for preparing parenteral formulations are known in the art and are described in REMINGTON; THE SCIENCE AND PRACTICE OF PHARMACY, supra, at pages 780-820. Parenteral compositions may be provided in a liquid formulation or as a solid which will be mixed with a sterile injectable medium shortly before administration. In an especially preferred embodiment, parenteral compositions are provided in unit dosage form for ease of administration and uniformity of dosage.

[0066] In accordance with the present invention in a further aspect there is provided a method of treating infertility comprising: (a) measuring the level of AMH in a subject's serum; and (b) administering to the subject a dose of, or a dose equivalent to, 1-24 µg, for example 2-24 µg, for example 2 to 15 µg, of human-derived recombinant FSH. Preferably the dose is, or is equivalent to, 4.5 to 12.5 µg, for example 5 to 12.5 µg, for example 6 to 12.5 µg, for example 6.3 to 12 µg, of Human-derived recombinant FSH.

[0067] In accordance with the present invention in a further aspect there is provided a method of treating infertility comprising: (a) determining (e.g. measuring) the level of AMH in a subject's serum; and (b) administering a dose of (e.g., daily), or dose equivalent to, 9 to 14 μg, e.g. 11 to 13 μg, e.g. 12 μg, human-derived recombinant follicle stimulating hormone (FSH) to one (o) subject having a serum AMH level of <15 pmol/L (e.g., 0.05 pmol/L to 14.9 pmol/L). Preferably the FSH is a recombinant FSH ("rFSH" or "recFSH"). Preferably the FSH is a human-derived recombinant FSH cell line. The dose provides an effective response while minimizing the risk of OHSS.

[0068] In accordance with the present invention in a further aspect there is provided a method of treating infertility comprising: (a) determining (e.g. measuring) the level of AMH in a subject's serum; and (b) administering a dose of (e.g., daily), or dose equivalent to, 5 to 12.5 μg of recombinant human-derived follicle stimulating hormone (FSH) to a (the) individual having a serum AMH level of >15 pmol/L. The dose (eg daily) may be, or be equivalent to, 6 to 10 μg of human-derived recombinant follicle stimulating hormone (FSH). Preferably the FSH is a recombinant FSH ("rFSH" or "recFSH"). Preferably the FSH is a human-derived recombinant FSH cell line. The dose provides an effective response while minimizing the risk of OHSS.

[0069] In one embodiment, the method includes a step of administering a dose of (e.g., daily), or dose equivalent to, 5 to 12 μg, e.g. 7 to 12 μg, e.g. 8.7 to 10 µg, human-derived recombinant FSH (preferably 9 to 10 µg human-derived recombinant FSH) to one (the) individual having a serum AMH level of 15 to 24.9 pmol/L in another embodiment, the method includes a step of administering a dose of (e.g., daily), or dose equivalent to, 5 to 12 µg of human-derived recombinant FSH (e.g., 7 to 12 µg, e.g. 6 to 9 µg, e.g., 7 to 8 µg, e.g. 7.3 to 8 µg human-derived recombinant FSH) to one (o) individual having a serum AMH level of 25 to 34.9 pmol/L. In another embodiment, the method includes a step of administering a dose of (e.g., daily), or dose equivalent to, 5 to 11 µg of human-derived recombinant FSH (e.g., 6 to 7 µg, e.g., 6, 3 to 7 μg, human-derived recombinant FSH) to one (the) individual having a serum AMH level of > 35 pmol/L.

[0070] In accordance with the present invention in a further aspect there is provided a method of treating infertility comprising: (a) determining (e.g. measuring) the level of AMH in a subject's serum; and (b) administering a dose of (e.g., daily), or dose equivalent to, 0.09 to 0.19 µg (e.g., 0.09 to 0.17 µg) of human-derived recombinant FSH per kg of subject's body weight, wherein the subject has a serum AMH level of >15 pmol/L. Preferably the FSH is a recombinant FSH ("rFSH" or "recFSH"). Preferably the FSH is a human-derived recombinant FSH cell line. The dose provides an effective response while minimizing the risk of OHSS.

[0071] In one embodiment, the method includes a step of administering a dose of (e.g., daily), or dose equivalent to, 0.14 to 0.19 µg of human-derived recombinant FSH (preferably 0.15 to 0.15 to 0.19 µg). 0.16 µg human-derived recombinant FSH) per kg of subject body weight, the subject having a serum AMH level of 15 to 24.9 pmol/L. In another embodiment, the method includes a step of administering a dose of (e.g., daily), or dose equivalent to, 0.11 to 0.14 µg of human-derived recombinant FSH (preferably 0.12 to 0.13 µg of human-derived recombinant FSH) per kg of subject body weight, the subject having a serum AMH level of 25 to 34.9 pmol/L. In another embodiment, the method includes a step of administering a dose of (e.g., daily), or dose equivalent to, 0.10 to 0.11 µg of human-derived recombinant FSH per kg of subject body weight, the subject having a serum AMH level of >35 pmol/L. Preferably the FSH is a recombinant FSH ("rFSH" or "recFSH"). Preferably the FSH is a human-derived recombinant FSH cell line. Said doses provide an effective response while minimizing the risk of OHSS.

[0072] In accordance with the present invention in a further aspect there is provided a method of treating infertility comprising: (a) determining (e.g. measuring) the level of AMH in a subject's serum; and (b) administering a dose of (e.g., daily), or dose equivalent to, 0.15 to 0.21 µg (e.g., 0.19 to 0.21 µg) of human-derived recombinant FSH per kg of subject's body weight, wherein the subject has a serum AMH level of <15 pmol/L.

[0073] The administration preferably comprises a daily dose of, or a daily dose equivalent to, the amount of FSH defined above and in the claims. The (daily) dose may be a starting dose (it may be reduced, increased, or maintained during treatment).

[0074] The method may be a method of treating infertility in the patient's (individual) first stimulation protocol. It will be noted that for additional stimulation cycles, doses may be adjusted according to current ovarian response in the first cycle.

[0075] In accordance with the present invention in a further aspect there is provided a method of treating infertility comprising: (a) determining (e.g. measuring) the level of AMH in a subject's serum;

[0076] and (b) if the subject has a serum AMH level of <15 pmol/L (eg, 0.05 pmol/L to 14.9 pmol/L), give the subject a dose of, or dose equivalent to, 10 to 14 μg, eg 11 to 13 μg, eg 12 μg, human-derived recombinant follicle stimulating hormone (FSH); or

[0077] If the subject has a serum AMH level of 15 to 24.9 pmol/L, administer to the subject a dose of, or a dose equivalent to, 0.14 to 0.19 μg of human-derived recombinant FSH ( preferably 0.15 to 0.16 µg human-derived recombinant FSH) per kg subject body weight; or

[0078] If the subject has a serum AMH level of 25 to 34.9 pmol/L pmol/L, administer to the subject a dose of, or a dose equivalent to, 0.11 to 0.14 μg of recombinant FSH derived of human (preferably 0.12 to 0.13 µg of human-derived recombinant FSH) per kg of subject body weight; or

[0079] If the subject has a serum AMH level of >35 pmol/L pmol/L, administer to the subject a dose of, or a dose equivalent to, 0.10 to 0.11 μg of human-derived recombinant FSH per kg of the individual's body weight.

[0080] For a patient (individual) having serum AMH of 5,014.9 pmol/L, a dose of, or equivalent dose of, 6 to 18 μg, e.g. 8 to 11 μg, e.g. 8.5 to 10.2 µg of human-derived recombinant FSH can be administered. For a patient (subject) having serum AMH 15.0-29.9 pmol/L, a dose of, or a dose equivalent to, 4.8 to 15 μg, e.g. 6 to 9 μg, e.g. 6 .8 to 8.5 µg of human-derived recombinant FSH can be administered. For a patient (subject) having serum AMH 30-44.9 pmol/L, a dose of, or a dose equivalent to, 3.6 to 12 μg, e.g. 4 to 7 μg, e.g. 5.1 to 6.8 µg of human-derived recombinant FSH can be administered. For a patient (subject) having serum AMH 45 pmol/L or more, a dose of, or a dose equivalent to, 2 to 9 μg, e.g. 2.4 to 9 μg (e.g., 3.4 to 5 .1 µg) or 2 to 5 µg of human-derived recombinant FSH can be administered. For a patient (subject) having serum AMH of 5 pmol/L or less, a dose of, or a dose equivalent to 7.2 to 24 μg, e.g. 10 to 15 μg e.g. 10.2 to 13, 6 μg, human-derived recombinant FSH can be administered. In some examples, a dose of, or a dose equivalent to, 4.8 to 18 µg, e.g. 6 to 11 µg, e.g. 6.8 to 10.2 µg, human-derived recombinant FSH is administered. Preferably the FSH is a recombinant FSH ("rFSH" or "recFSH"). Preferably the FSH is a human-derived recombinant FSH cell line. Administration preferably comprises a daily dose of, or a daily dose equivalent to, the amount of FSH defined above and in the claims. The (daily) dose may be a starting dose (it may be reduced, increased, or maintained during treatment).

Detailed description of the invention

[0081] The present invention will now be described in more detail with reference to the accompanying drawings, in which:

[0082] Figure 1 shows a plasmid map of the pFSHalpha/beta expression vector;

[0083] Figure 2 shows the α2,3-sialyltransferase (ST3GAL4) expression vector;

[0084] Figure 3 shows the α2,6-sialyltransferase expression vector (ST6GAL1);

[0085] Figure 4 shows % abundance sialic acid distribution of examples of recombinant FSH produced by PER.C6® cells stably expressing FSH after genetic engineering with α2,3-sialyltransferase;

[0086] Figure 5 shows % abundance of glycan charge distribution of examples of recombinant FSH produced by PER.C6® cells stably expressing FSH after genetic engineering with α2,3-sialyltransferase;

[0087] Figure 6 shows a comparison of inhibin-B concentration following administration of 225IU Gonal f (bottom line, dotted line) and 225 IU Example (top line, solid line) of the present invention;

[0088] Figure 7 shows the effect of body weight on oocytes retrieved in the low AMH treatment group (Example 10.10A); and

[0089] Figure 8 shows the effect of body weight on recovered oocytes in the high AMH treatment group.

Sequence Selection Human FSH

[0090] The gene coding region for the FSH alpha polypeptide was used according to Fiddes and Goodman. (1981). The sequence is grouped as AH007338 and at the time of construction there were no other variants of said protein sequence. The sequence is referred to herein as SEQ ID NO:1.

[0091] The gene coding region for the FSH polypeptide beta was used according to Keene et al (1989). The sequence is grouped as NM_000510 and at the time of construction there were no other variants of said protein sequence. The sequence is referred to herein as SEQ ID NO: 2

Sialyltransferase

[0092] α2,3-Sialyltransferase - The coding region of the gene for beta-galactoside alpha-2,3-sialyltransferase 4 (α2,3-sialyltransferase,ST3GAL4) was used according to Kitagawa and Paulson (1994). The sequence is grouped as L23767 and referred to herein as SEQ ID NO: 3.

[0093] α2,6-Sialyltransferase - The gene coding region for beta-galactosamide alpha-2,6-sialyltransferase 1 (α2,6-sialyltransferase, ST6GAL1) was used according to Grundmann et al. (nineteen ninety). The sequence is grouped as NM_003032 and referred to herein as SEQ ID NO: 4.

EXAMPLES

[0094] Example 1 Construction of the FSH expression vector

[0095] A coding sequence of FSH alpha polypeptide (AH007338, SEQ ID NO: 1) and FSH beta polypeptide (NM_003032, SEQ ID NO: 2) was amplified by PCR using primer combinations FSHa-fw and FSHa- rev and FSHb-fw and FSHb-rec respectively.

[0096] FSHa-fw 5'-CCAGGATCCGCCACCATGGATTACTACAGA AAAATATGC-3' (SEQ ID NO:9)

[0097] FSHa-rev 5'-GGATGGCTAGCTTAAGATTTGTGATAATAA C-3' (SEQ ID NO:10)

[0098] FSHb-fw 5'-CCAGGCGCGCCACCATGAAGACACTCCAG TTTTTC-3' (SEQ ID NO: 11)

[0099] FSHb-rev 5'-CCGGGTTAACTTATTATTCTTTCATTTCAC CAAAGG-3' (SEQ ID NO: 12)

[00100] The resulting amplified beta FSH DNA was digested with restriction enzymes AscI and HpaI and inserted into the AscI and HpaI fields in the CMV-oriented mammalian expression vector carrying a neomycin selection marker. Similarly, the alpha FSH DNA was digested with BamHI and NheI and inserted into the BamHI and NheI fields in the expression vector already containing the FSH DNA beta polypeptide.

[00101] Vector DNA was used to transform E.coli strain DH5α. Colonies were picked up for amplification. Colonies containing the vector containing both alpha and beta FSHs were selected for sequencing and all contained the correct sequences according to SEQ ID NO: 1 and SEQ ID NO: 2. Plasmid pFSH A+B#17 was selected for transfection ( Figure 1).

[00102] Example 2 Construction of the expression vector ST3

[00103] The coding sequence for beta-galactoside alpha-2,3-sialyltransferase 4 (ST3, L23767, SEQ ID NO: 3) was amplified by PCR using the primer combination 2,3STfw and 2,3STrev.

[00104] 2,3STfw 5'-CCAGGATCCGCCACCATGTGTCCTGCAGG CTGGAAGC-3' (SEQ ID NO: 13)

[00105] 2,3STrev 5'-TTTTTTTTCTTAAGTCAGAAGGACGTGAGGT TCTTG-3' (SEQ ID NO: 14)

[00106] The resulting amplified ST3 DNA was digested with restriction enzymes BamHI and AflII and inserted into the BamHI and AflII fields in the CMV-oriented mammalian expression vector carrying a hygromycin resistance marker. The vector was amplified as previously described and sequenced. Clone pST3#1 (Figure 2) contained the correct sequence according to SEQ ID NO: 3 and was selected for transfection.

[00107] Example 3 Construction of the expression vector ST6

[00108] The coding sequence for beta-galactosamide alpha-2,6-sialyltransferase 1 (ST6, NM_003032, SEQ ID NO: 4) was amplified by PCR using the primer combination 2.6STfw and 2.6STrev.

[00109] 2,6STfw 5'-CCAGGATCCGCCACCATGATTCACACCAA CCTGAAG-3' (SEQ ID NO: 15)

[00110] 2,6STrev 5'-TTTTTTTTCTTAAGTTAGCAGTGAATGGTCC GG-3' (SEQ ID NO: 16)

[00111] The resulting amplified ST6 DNA was digested with restriction enzymes BamHI and AflII and inserted into the BamHI and AflII fields in the CMV-oriented mammalian expression vector carrying a hygromycin resistance marker. The vector was amplified as previously described and sequenced. Clone pST6#11 (Figure 3) contained the correct sequence according to SEQ ID NO: 4 and was selected for transfection.

[00112] Example 4 Stable expression of pFSH α+β in PER.C6® cells. Isolation of transfection and reading of clones.

[00113] FSH producing PER.C6® clones were generated by expression of both FSH polypeptide chains from a single plasmid (see Example 1).

[00114] To obtain stable clones a liposome-based transfection agent with the pFSH α+β construct. Stable clones were selected in VPRO supplemented with 10% FCS and containing G418. Three weeks after transfection, G418 resistant clones developed. Clones were selected for isolation. Isolated clones were grown in selection medium until 70-80% confluent. Supernatants were tested for FSH protein content using an FSH selective ELISA and pharmacological activity at the FSH receptor in the cloned cell line using a cAMP accumulation test. Clones expressing functional protein were progressed to expansion of culture in 24-well, 6-well and T80 flasks.

[00115] Studies to determine productivity and material quality from seven clones were initiated in T80 flasks to generate sufficient material. Cells were cultured in supplemented medium as previously described for 7 days and the supernatant collected. Productivity was determined using the FSH selective ELISA. The isoelectric profile of the material was determined by isoelectric focusing (IEF) by methods known in the art. Clones with sufficient productivity and quality were selected for sialyltransferase genetic engineering.

[00116] Example 5 Level of sialylation is increased in cells that overexpress α2,3-sialyltransferase. Stable expression of pST3 in FSH expressing PER.C6® cells; isolation of transfection and clone reading.

[00117] PER.C6® clones producing highly sialylated FSH were generated by the expression of α2,3 sialyltransferase from separate plasmids (Example 2) in PER.C6® cells that already express both FSH polypeptide chains (the from Example 4). Clones produced from PER.C6® cells as determined in Example 4 were selected for their characteristics including productivity, good developmental profile, production of functional protein, and produced FSH that included some sialylation. Stable clones were generated as previously described in Example 4. Clones were isolated, expanded and tested. The α2,3-sialyltransferase clones were adapted to serum-free medium and suspension conditions.

[00118] As before, clones were tested using FSH selective ELISA, functional response in an FSH receptor cell line, IEF, metabolic clearance coefficient and Steelman Pohley analysis. The results were compared to a commercially offered recombinant FSH (Gonal-f, Serono) and to parental PER.C6® FSH cell lines. FSH produced by most clones showed significantly improved sialylation (ie, on average more FSH isoforms with high numbers of sialic acids) compared to FSH expressed without α2,3-sialyltransferase. In conclusion, expression of FSH together with sialyltransferase in PER.C6® cells resulted in increased levels of sialylated FSH compared to cells expressing only FSH.

[00119] Example 6 Production and Purification Overview

[00120] A procedure was developed to produce FSH in PER.C6® Cells that were cultured in suspension in serum-free medium. The procedure is described below and has been applied to several cell lines of PER.C6® producing FSH.

[00121] FSH from α2,3-clone (Example 5) was prepared using a modification of the method described by Lowry et al. (1976).

[00122] For the production of PER.C6®-FSH, cell lines were adapted to a serum-free medium, ie Excel 525 (JRH Bios-ciences). Cells were first grown to form a 70%-90% confluent monolayer in a T80 culture flask. On passage the cells were resuspended in serum free medium, Excel 525 + 4 mM L-Glutamine, at a cell density of 0.3 x 10 6 cells/ml. A 25 ml cell suspension was placed in a 250 ml shake flask and shaken at 100 rpm at 37°C in 5% CO 2 . After reaching a cell density of > 1 x 10 6 cells/mL, cells were subcultured to a cell density of 0.2 or 0.3 x 10 6 cells/mL and further cultured in shake flasks at 37°C. °C, 5% CO2 and 100 rpm.

[00123] For FSH production, cells were transferred to a serum-free production medium, i.e. VPRO (JRH Biosciences), which supports the growth of PER.C6® Cells to very high cell densities (generally > 107 cells/mL in a batch culture). Cells were first grown at >1 x 10 6 cells/ml in Excel 525, then centrifuged for 5 min at 1000 rpm and subsequently suspended in VPRO medium + 6 mM L-glutamine at a density of 1 x 10 6 cells/ml. Cells were then cultured in a shaker flask for 7-10 days at 37°C, 5% CO2 and 100 rpm. During this period, cells developed to a density of > 107 cells/mL. Culture medium was collected after cell viability began to decline. Cells were centrifuged for 5 min at 1000 rpm and the supernatant was used for FSH quantification and purification. FSH concentration was determined using ELISA (DRG EIA 1288).

[00124] Subsequently, the purification of FSH was performed using modification of the method described by Lowry et al. (1976). Purification using selective charge chromatography was performed to enrich the highly sialylated forms by methods well known in the art.

[00125] During all chromatographic procedures, the enrichment of the sialylated forms of FSH as claimed herein was confirmed by RIA (DRG EIA 1288) and/or IEF.

[00126] Example 7 Quantification of Relative Amounts of α2.3 and α2.6 Sialic Acid

[00127] The relative percentage amounts of α2.3 and α2.6 sialic acid in purified rFSH (Example 6) were measured using known techniques.

[00128] N-Glycans were released from the samples using PNGase F under denaturing conditions and then labeled with 2-aminobenzamide. The released glycan forms were then separated and analyzed by a Weak Anion Exchange (WAX) column to determine charge distribution. Labeled glycans treated with 2,3,6,8 sialidase for determination of total sialic acid and 2,3 sialidase for determination of 2,3 sialic acid, were further analyzed by wax column.

[00129] The relative percentages of charged glycans were calculated from the structures present in the undigested and digested glycan groups and are shown in Figure 4 (for 8 samples). Referrals have been observed to be in the ranges 50% - 70% (e.g. about 60% or 65%) for α2,3 sialylation and 28 to 50%, generally 30 to 35% (e.g. about 31% or 35%), for α2.6 sialylation.

[00130] Example 8 Quantification of Relative Amounts of sialylated mono, di, tri and tetra glycan structures

[00131] The relative percentage amounts of mono, di, tri and tetra sialylated structures in glycans extracted from purified rFSH (Example 6) were measured using known techniques.

[00132] N Glycans were released from the samples using PNGase F under denaturing conditions and then labeled with 2-aminobenzamide. Glycans were released from the samples using PNGase F under denaturing conditions and then labeled with 2-aminobenzamide. The released glycan forms were then separated and analyzed by a Weak Anion Exchange (WAX) column to determine the sialylation distribution. The relative amounts of neutral, mono-sialylated, di-sialylated, tri-sialylated and tetra-sialylated structures are shown in Figure 5 (for the 8 samples shown in Figure 4).

[00133] rFSH includes neutral, monosialylated, disialylated, trisialylated and tetrasialylated glycan structures with relative amounts as follows: 5-6% neutral; 15-17% monosialylated; 26-30% di--sialylated; 30-32% tri-sialylated and 17-23% tetra-sialylated.

[00134] Example 8A

[00135] The relative percentage amounts of α2,6 sialic acid in purified rFSH extracted from nine samples of purified rFSH (produced by the methods of Example 6) were measured using known techniques.

[00136] N-Glycans were released from the samples using PNGase F under denaturing conditions and then labeled with 2-aminobenzamide. The released glycan forms were then separated and analyzed by a Weak Anion Exchange (WAX) column to determine charge distribution. Labeled glycans treated with 2,3,6,8 sialidase for determination of total sialic acid and 2,3 sialidase for determination of 2,3 sialic acid, were further analyzed by wax column (see Example 8). The analysis allows the calculation of α2,6 sialic acid.

[00137] The relative percentages of charged glycans were calculated from structures present in the undigested and digested glycan groups and are shown in the following table. Referrals were observed to be in a range of 25 to 50%, generally 30 to 35% for α2,6 sialylation.

[00138] Relative percent bisection amounts of Glc-Nac, GalNac and 1-Fucose Lewis in glycans extracted from nine samples of purified rFSH (produced by the methods of Example 6) were measured using known techniques. N-Glycans were released from the glycoprotein using PNGase F and labeled with 2-aminobenzamide (2AB). Analysis was performed by two-dimensional (2D) HPLC analysis in combination with enzymatic degradation of glycans. For verification, glycans were analyzed by MALDI-MS. The relative amounts of alpha 2,6-sialic acid and terminal residues are shown in the following table, along with those for Gonal F (CHO cell-derived recombinant FSH) and Bravelle (human urinary FSH).

[00139] 1 Value of 3.1 is total ^ Fucose Lewis. 2 Not determined.

[00140] It can be seen that the amount of GalNac in the FSH of the present invention varies between about 44.9 and 51%, average of about 47.1%.

[00141] It can be seen that the amount of bisection of Glc-Nac in the FSH of the present invention varies between 8.7 and 13.9%, average of approximately to 10.9%.

[00142] It can be seen that the amount of 1 Fucose Lewis in the FSH of the present invention varies between 161 and 23.3%, average approximately to 19%.

[00143] It can be seen that the amount of 2 Fucose Lewis in the FSH of the present invention varies between 1.9 and 4.4%, average approximately to 3.7%.

[00144] Example 9 - A multiple dose study investigating the safety, tolerability, pharmacokinetics, pharmacodynamics, and immunogenicity of FE 999049 compared to GONAL-F.

Study population

[00145] A total of 48 (24 on each drug) healthy women received daily doses of 14.6 µg of FE 999049 (the composition according to the present invention, produced according to Example 6) or 16.5 µg of Gonal -F for seven days.

Security Results

[00146] Multiple dose administration of FE 999049 and GONAL-F was safe and generally well tolerated as assessed by Adverse Events (AEs), vital signs, ECG, clinical laboratory measurements, and physical examination. No serious adverse effects or death occurred during the study.

Pharmacokinetics Results

[00147] Following administration of FE 999049 and GONAL-F for 7 days, FSH concentration values as assessed immediately before the next injection increased and appeared to reach a steady-state level after 6-7 days. However the exposure (AUC and Cmax) of FE 999049 was 60% higher compared to Gonal-F.

Pharmacodynamics Results

[00148] Concentrations of inhibin-B (see Figure 6), oestradiol, and progesterone all increased subsequent to administration of FE 999049 and GONAL-F, however to a greater extent following administration of FE 999049 compared to GONAL-F . not only the number but also the size distribution of follicles showed a greater response to FE 999049 compared to GONAL-F.

[00149] Example 9 demonstrates that FSH having a specific amount (17-23%) of tetrasialylated glycan structures and for example specific amounts of α2,3 sialylation and α2,6 sialylation is markedly more potent than the recombinant FSH products that are currently on the market.

[00150] Example 10 - A multiple dose study investigating FE 999049 compared to GONAL-F.

[00151] The following describes groups of randomized, controlled, adjunct-blind, multinational, multicenter, parallel trials evaluating the dose-response relationship of EF 999049 in patients undergoing controlled ovarian stimulation for in vitro fertilization (IVF) / intracytoplasmic sperm injection (ICSI). The patient population was 265 IVF patients aged 18 to 37 years, with BMI 18.5 to 32.0 kg/m2.

[00152] The test is designed as a dose response test with the number of oocytes retrieved as the primary endpoint. Secondary endpoints will explore the qualitative and quantitative impact of different doses of FE 999049 with respect to endocrine profile, follicular development, oocyte fertilization, embryo quality, and treatment efficiency (i.e., total and stimulation duration). The test is designed to assess the effectiveness of FE 999049 in establishing pregnancy when used in controlled ovarian stimulation for IVF/ICSI cycles.

[00153] Subjects were evaluated within 3 months prior to randomization to meet inclusion and exclusion criteria, including an anti-Müllerian hormone (AMH) assessment to increase the homogeneity of the test population with respect to response and minimize the number of poor and hyper potential responders to the doses of FE 999049 and dose of GONAL-F used in the test. AMH assessment was measured using the AMH Gen-II enzyme-linked immunosorbent assay kit (Beckman Coulter, Inc., Webster, Texas). Said test can detect AMH concentrations greater than 0.57 pmol/L with a lower limit of quantification of 1.1 pmol/L.

[00154] On day 2-3 of their menstrual cycle, subjects were randomized in a 1:1:1:1:1:1 mode to treatment with either 90 IU, 120 IU, 150 IU, 180 IU or 210 IU FE 999049 or 150 IU GONAL-F, and ovarian stimulation started. Randomization was stratified according to AMH level in readout [5.0-14.9 pmol/L (low AMH) and 15.0 to 44.9 pmol/L (high AMH)).

[00155] Gonal-F is filled by mass (FbM) on FDA request; reference to μg dose is therefore appropriate. The Gonal -F label indicates 600 IU/44 μg, which indicates that 150 IU is 11 μg. However, there is some variation and the batch certificate for this test indicated that 11.3 μg of Gonal-F was equivalent to 150 IU. FE999049 doses are presented by protein content (μg) rather than biological activity. So the doses of FE999049 were 5.2 μg (90 IU), 6.9 μg (120 IU), 8.6 μg (150 IU), 10.3 μg (180 IU) or 12.1 μg (210 IU) .

[00156] Subject and dose distribution is determined as follows (data are subject numbers): Table 1

[00157] The daily dose level of FE 999049 or GONAL-F is fixed throughout the stimulation period. During stimulation, subjects are monitored on stimulation day 1, 4, and 6 and thereafter at least every other day. When 3 follicles >15 mm are observed, visits are performed daily. Subjects are treated with FE 999049 or GONAL-F for a maximum of 16 days.

[00158] To prevent a premature surge of LH, a GnRH antagonist (ganirelix acetate, ORGALUTRAN, MSD / Schering-Plough) can be started on day 6 of stimulation at a daily dose of 0.25 mg and continued through the period of stimulation. The triggering of final follicular maturation is performed on the day when >3 follicles with a diameter of >17 mm are observed. If there are <25 follicles with a diameter of >12 mm, 250 μg recombinant hCG (choriogonadotropin alfa, OVITRELLE, Merck Serono / EMD Serono) is administered. If there are 2535 follicles with a diameter >12 mm, 0.2 mg of GnRH agonist (triptorelin aceetate, DECAPEPTIL / GONAPEPTIL, Ferring Pharmaceuticals) is administered. In case of excessive ovarian response, defined as >35 follicles with a diameter of >12 mm, treatment is cancelled. In the event of a poor ovarian response, defined as <3 follicles with a diameter >10 mm observed on day 10 of stimulation, the cycle may be cancelled.

[00159] Oocyte retrieval occurs 36h (± 2h) after triggering final follicular maturation and oocytes inseminated by IVF and/or ICSI. Fertilization and embryo development are assessed from oocyte retrieval to the day of transfer. For subjects who underwent final follicular maturation triggering with hCG, a best quality blastocyst available is transferred on day 5 after oocyte retrieval while the remaining blastocysts are frozen. For individuals who undergo final follicular maturation triggering with GnRH agonist, no embryo transfer occurs in the new cycle and blastocysts are instead frozen at day 5. Vaginal progesterone tablets (LUTINUS, Ferring Pharmaceuticals) 100 mg 3 times daily are provided for luteal phase support from the day after oocyte retrieval until the day of the pregnancy clinic visit. A βhCG test is performed 13-15 days after embryo transfer and clinical pregnancy will be confirmed by transvaginal ultrasound (TVU) 5-6 weeks after embryo transfer.

Results

[00160] The number of oocytes retrieved (major endpoint) is shown in the following table.Table 2

[00161] Data are averages (SD)

[00162] The main objective was achieved: a significant dose-response relationship was established for EF 999049 with respect to the number of oocytes retrieved. This finding was observed not only for the theotal test population, but also for each of the two AMH strata used in randomization.

[00163] A significant dose response for FE 999049 was demonstrated for all key pharmacodynamic endpoints, eg estradiol, inhibin B and inhibin A. At a similar microgram dose level, pharmacodynamic responses with FE 999049 were higher than with GONAL-F (referred results are not shown).

[00164] Serum FSH concentrations after exposure to FE 999049 were significantly higher than for GONAL-F. The results confirm that the PK profile of FE 999049 differs from that of GONAL-F.

[00165] Fertilization coefficients, blastocyst development and pregnancy coefficients in IVF/ICSI patients treated with FE 999049 were within expectations.

[00166] There were no safety concerns with the use of FE 999049. Good local tolerability has been documented.

Additional Analysis

[00167] Applicants further analyzed the data to identify the dose(s) of FE 999049 that met all of the following criteria with respect to the number of oocytes retrieved:

[00168] Oocytes recovered in range 8-14

[00169] Minimize proportion of patients with <8 oocytes

[00170] Minimize proportion of patients with >20 oocytes

[00171] Applicants also investigated the impact of body weight. If relevant, the dose is converted to μg/kg for an average subject. The referred value of μg/kg and ±0.01 μg/kg is evaluated in a model with respect to the distribution of retrieved oocytes as well as the safety profile, and the optimal dose is identified.

Lower stratum of AMH

[00172] As seen in Table 2, the dose of FE999049 that met the first criterion (Oocytes retrieved in a range of 8-14) was 12.1 μg (mean 9.4 oocytes retrieved). The distribution of oocytes is shown in Table 3 below.Table 3

[00173] Data are % of individuals

[00174] As shown by the box and arrow, the 12.1 μg dose of FE999049 provides recovery of the most desirable number of oocytes in 60% of subjects in the low AMH group. This is a significant improvement in Gonal-F (most desirable number of oocytes in only 33% of subjects).

[00175] Table 4 below shows the analysis of excessive response signals in the low AMH stratum (data are numbers of individuals). It can be seen that there were no indications of early OHSS of a moderate or severe nature and there were no incidents of preventive action being necessary; there was no concern associated with the 12.1 μg dose of FE999049 in a patient having low AMH. Table 4

[00176] Figure 7 shows the effect of body weight on recovered oocytes (for the low AMH stratum), for the various doses. Arrows indicate the number of oocytes recovered from subjects of body weight between 45 kg and 90 kg treated with the 12.1 μg dose. As can be seen (text box) the variation between patients weighing 45kg and those weighing 90kg is less than around 0.5 oocytes; in other words, body weight dosing is not necessary in patients with low AMH when the dose of FE999049 is at least 12 μg, as there is not a significant change in oocytes recovered with body weight at that dose.

[00177] Thus Applicants have noted that a dose of, or equivalent dose of, 6 to 18 µg, e.g. 9 to 14 µg, e.g. 12 µg, of human-derived recombinant FSH is suitable for use in the treatment of infertility in a patient having serum AMH <15 pmol/L, eg 0.05-14.9 pmol/L eg 5.0-14.9 pmol/L. The dose provides an effective response while minimizing the risk of OHSS.

High AMH Stratum

[00178] As seen in Table 2, three doses of FE999049 fulfilled the first criterion (oocytes recovered in a range of 8-14): 6.9 μg (mean of 9.1 oocytes recovered), 8.6 μg (mean of 10, 6 oocytes recovered), and 10.3 μg (mean of 13.6 oocytes recovered).

[00179] Figure 8 shows the effect of body weight on recovered oocytes (for the high AMH stratum), for the various doses. Arrows indicate the number of oocytes recovered from subjects of body weight between 45 kg and 90 kg treated at doses of 6.9 µg, 8.6 µg and 10.3 µg. As can be seen (text boxes) the variation is significant: for the 6.9 μg dose 6 additional oocytes will be recovered from a 45 kg patient compared to a 90 kg patient; for the 8.6 μg dose 4 additional oocytes will be retrieved from a 45 kg patient compared to a 90 kg patient; and for the 10.1 μg dose 2.5 additional oocytes will be retrieved from a 45 kg patient compared to a 90 kg patient. In other words, body weight dosing has an impact in patients with high AMH when the dose of FE999049 is less than 12 μg, due to the fact that there is a significant variation in oocytes recovered with body weight at said doses.

[00180] Table 5a below shows an additional rupture of oocytes retrieved (from Table 2) by AMH. This shows the doses that met the first criterion (oocytes retrieved in a range of 8-14) for each AMH substratum (boxes).Table 5a

[00181] Table 5b below shows the analysis of patients where treatment was canceled due to either excessive response or agonist triggering, for those subgroups. For example, one patient in AMH stratum of 25-34 pmol/L canceled due to excessive response following the 10.3 μg dose and one patient in AMH styrate of 25-34 pmol/L canceled due to excessive response following the 12.1 μg dose; one patient on estyratod and AMH of 35-45 pmol/L canceled after triggering the agonist after the 10.3 μg dose; and one patient in the AMH stratum of 35-45 pmol/L subsequently canceled agonist triggering following the 6.9 μg dose.Table 5b

[00182] It can be seen therefore that when making the dose by body weight (Fig 8) and AMH level is useful in the high AMH stratum, to minimize cancellations and maximize oocyte recovery.

[00183] Applicants have observed that the following doses provide an effective response while minimizing the risk of OHSS (kg is kg of patient body weight).

[00184] The following is appropriate if dosing by body weight is not desired.

[00185] The following is appropriate if fewer AMH categories are required.

[00186] The following is appropriate if dosing by body weight is not desired. .

[00187] Thus Applicants have observed that a dose of, or a dose equivalent to, 9 to 14 μg, e.g. 12 μg, of human-derived recombinant FSH is suitable for use in the treatment of infertility in a patient having serum AMH < 15 pmol/L eg 0.0514.9 pmol/L eg 5.0-14.9 pmol/L. The dose provides an effective response while minimizing the risk of OHSS.

[00188] Applicants have noted that a dose of, or a dose equivalent to, 5 to 12.5 µg, e.g. 6 to 10.5 µg, of human-derived recombinant FSH is suitable for use in treating infertility in a patient having serum AMH >15 pmol/L. The dose provides an effective response while minimizing the risk of OHSS.

[00189] Applicants have noted that a dose of (eg, daily), or dose equivalent to, 0.09 to 0.19 μg of human-derived recombinant FSH per kg of patient body weight is suitable for use in the infertility treatment in a patient having a serum AMH level of >15 pmol/L. Applicants have observed that a dose of (e.g., daily), or dose equivalent to, 0.14 to 0.19 µg of human-derived recombinant FSH (preferably 0.15 to 0.16 µg of human-derived recombinant FSH) per kg of patient body weight is suitable for use in treating infertility in a patient having a serum AMH level of 15 to 24.9 pmol/L. Applicants have observed that a dose of (e.g., daily), or dose equivalent to, 0.11 to 0.14 µg of human-derived recombinant FSH (preferably 0.12 to 0.13 µg of recombinant FSH derived from human) per kg of patient body weight is suitable for use in treating infertility in a patient having a serum AMH level of 25 to 34.9 pmol/L. Applicants have noted that a dose of (e.g., daily), or dose equivalent to, 0.10 to 0.11 µg of human-derived recombinant FSH per kg of patient body weight is suitable for use in the treatment of infertility in a patient having a serum AMH level of > 35 pmol/L. Said doses provide an effective response while minimizing the risk of OHSS.

[00190] Applicants have observed that a dose of (e.g., daily), or dose equivalent to, 0.15 to 0.21 μg (e.g., 0.16 μg) of human-derived recombinant FSH per kg of body weight of the patient is suitable for use in treating infertility in a patient having a serum AMH level of <15 pmol/L, for example, for the first cycle of stimulation with human-derived recombinant FSH. However, it is not necessary for patients to be dosed by body weight at this AMH level.

[00191] Example 10 A - Individualized COS Protocol (low AMH)

[00192] Selected patients are to undergo COS for in vitro fertilization (IVF) / intracytoplasmic sperm injection (ICSI) by methods known in the art. The pretreatment protocol includes assessment/reading of AMH in the patient's serum using the AMH Gen-II Enzyme Linked Immunosorbent Test Kit (Beckman Coulter, Inc., Webster, Texas). This test can detect AMH concentrations greater than 0.57 pmol/L with a lower limit of quantification of 1.1 pmol/L. AMH can be measured using other Test kits (eg available from Roche).

[00193] The COS protocol proceeds in the usual way regardless of the administration of the initial dose of FE 999049 according to the AMH level in the reading. A patient with an AMH level of <14.9 pmol/L would be given an initial daily dose of approximately 12 μg FE 999049, a human-derived recombinant FSH product manufactured according to the method of Example 6. A patient with an AMH level of 15 to 24.9 pmol/L would receive a daily starting dose of 0.15 to 0.19 μg of the human-derived recombinant FSH per kg of patient body weight. A patient with an AMH level of 25 to 34.9 pmol/L would receive a daily starting dose of 0.11 to 0.13 μg of the human-derived recombinant FSH per kg of patient body weight. A patient with an AMH level of > 35 pmol/L would receive a daily starting dose of 0.10 to 0.11 μg of the human-derived recombinant FSH per kg of the patient's body weight.

[00194] Example 11 - Individualized COS Protocols.

[00195] The doses in this protocol are less preferred than in Example 10A.

[00196] Selected patients are to undergo COS for in vitro fertilization (IVF) / intracytoplasmic sperm injection (ICSI) by methods known in the art. The pretreatment protocol includes assessment/reading of AMH in the patient's serum using the AMH Gen-II Enzyme Linked Immunosorbent Test Kit (Beckman Coulter, Inc., Webster, Texas). This test can detect AMH concentrations greater than 0.57 pmol/L with a lower limit of quantification of 1.1 pmol/L.

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Increased biological activity due to basic isoforms in recombinant human follicle-stimulating hormone produced in a human cell line. J. Clin. Endocrinol. Metab., 79, 756-760 Fox KM, Dias JA, and Van Roey P. (2001). Threedimensional structure of human follicle-stimulating hormone. Mol Endocrinol. 15(3), 378-89 Grabenhorst E, Hoffmann A, Nimtz M, Zettlmeissl G, and Conradt HS. (1995). Construction of stable BHK-21 cells coexpressing human secretory glycoproteins and human Gal(beta 1-4)GlcNAc-R alpha 2,6-sialyltransferase alpha 2,6-linked NeuAc is preferentially attached to the Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-3)-branch of diantennary oligosaccharides from secreted recombinant beta-trace protein. Eur J Biochem. 232(3), 718-25. Green ED and Baenziger JU. (1988). Asparagine-linked oli-gosaccharides on lutropin, follitropin, and thyrotropin. II. Distributions of sulfated and sialylated oligosaccharides on bovine, ovine, and human pituitary glycoprotein hormones. J Biol Chem. 263(1), 36-44. Grundmann,U., Nerlich,C., Rein,T. and Zettlmeissl, G. (1990). Complete cDNA sequence encoding human beta-galactoside alpha-2,6-sialyltransferase. G Nucleic Acids Res. 18 (3), 667 Howles, C.M. (1996). Genetic engineering of human FSH (Gonal-F). Hum Reprod. Update, 2,172-191. Kagawa Y, Takasaki S, Utsumi J, Hosoi K, Shimizu H, Ko- chibe N, and Kobata A. (1988). Comparative study of the asparagine- linked sugar chains of natural human interferon-beta 1 and recombinant human interferon-beta 1 produced by three different mammalian cells. J Biol Chem. 263(33), 17508-17515. Keene, J.L., Matzuk, M.M., Otani, T., Fauser, B,C,J,M., Galway, A.B., Hsueh, A.J.W. and Boime, I. (1989). Expression of Bio-logically active Human Follitropin in Chinese Hamster Ovary Cells. The Journal of Biological Chemistry, 264(9), 4769-4775. Kitagawa,H. and Paulson,J.C (1994) Cloning of a novel alpha 2,3-sialyltransferase that sialylates glycoprotein and glycolipid car-bohydrate groups. J. Biol. Chem. 269(2), 1394-1401. Lee EU, Roth J, and Paulson JC (1989) Alteration of terminal glycosylation sequences on N-linked oligosaccharides of Chinese hamster ovary cells by expression of beta-galactoside alpha 2,6- sialyltransferase. J Biol Chem. 264(23), 13848-13855. de Leeuw, R., Mulders, J., Voortman, G. Rombout, F. Damm, J. and Kloosterboer, L. (1996) Structure-function relationship of recombinant follicle stimulating hormone (Puregon). Mol. Hum. Reprod., 2, 361-369. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. (1951) Protein measurement with the Folin phenol reagent. J Biol Chem. 193(1), 265-75. Lowry, PJ, McLean, C, Jones RL and Satgunasingam N. (1976) Purification of anterior pituitary and hypothalamic hormones Clin Pathol Suppl (Assoc Clin Pathol). 7, 16-21. Olivennes F, Howles CM, Borini A, Germond M, Trew G, Wikland M, Zegers-Hochschild F, Saunders H (2009) Individualizing FSH dose for assisted reproduction using a novel algorithm: the CONSORT study. Reprod Biomed Online. 2009 Feb;18(2): 195-204. Pierce JG, and Parsons TF (1981) Glycoprotein hormones: structure and function Annu Rev Biochem. 50, 465-495. Pricer WE Jr, and Ashwell G. (1971). The binding of desialylated glycoproteins by plasma membranes of rat liver. J Biol Chem. 246(15), 4825-33. Rathnam P, and Saxena BB. (1975). Primary amino acid sequence of follicle-stimulating hormone from human pituitary glands. I. alpha subunit. J Biol Chem.;250(17):6735-6746. Regoeczi E, Debanne MT, Hatton MC, and Koj A. (1978) Elimination of asialofetuin and asialoorosomucoid by the intact rat. Quantitative aspects of the hepatic clearance mechanism. Biochim Bi-ophys Acta. 541(3), 372-84. Royle L, Radcliffe CM, Dwek RA and Rudd PM (2006) Methods in Molecular Biology, ed I Brockhausen-Schutzbach (Humana Press), 347: Glycobiology protocols, 125-144. Ryan RJ, Keutmann HT, Charlesworth MC, McCormick DJ, Milius RP, Calvo FO and Vutyavanich T. (1987). Structure-function re-lationships of gonadotropins. Recent Prog Horm Res.;43,:383-429. Saxena BB and Rathnam P. (1976) Amino acid sequence of the beta subunit of follicle-stimulating hormone from human pituitary glands. J Biol Chem. 251(4), 993-1005 Steelman SL, and Pohley FM. (1953) Assay of the follicle stimulating hormone based on the augmentation with human chorionic gonadotropin. Endocrinology. 53(6), 604-616. Steer CJ, and Ashwell G. (1980) Studies on a mammalian hepatic binding protein specific for asialoglycoproteins. Evidence for receptor recycling in isolated rat hepatocytes. J Biol Chem. 255(7), 3008-13. Svensson EC, Soreghan B, and Paulson JC. (1990) Organ-ization of the beta-galactoside alpha 2,6-sialyltransferase gene. Evidence for the transcriptional regulation of terminal glycosylation. J Biol Chem. 265(34):20863-20868. Takeuchi M, Takasaki S, Miyazaki H, Kato T, Hoshi S, Ko- chibe N, and Kobata A (1988). Comparative study of the asparagine- linked sugar chains of human erythropoietins purified from urine and the culture medium of recombinant Chinese hamster ovary cells. J Biol Chem. 263(8), 3657-3663. Timossi CM, Barrios de Tomasi J, Zambrano E, González R, Ulloa-Aguirre A. (1998). A naturally occurring basically charged human follicle-stimulating hormone (FSH) variant inhibits FSH-induced androgen aromatization and tissue-type plasminogen activator enzyme activity in vitro. Neuroendocrinology. 67(3), 153-163. Timossi CM, Barrios-de-Tomasi J, González-Suárez R, Ar- ranz MC, Padmanabhan V, Conn PM, and Ulloa-Aguirre A. (2000). Dif-ferential effects of the charge variants of human follicle-stimulating hormone. J Endocrinol. 165(2), 193-205. Ulloa-Aguirre, A., Espinoza, R., Damian-Matsumura, P. and Chappel, S.C. (1988) Immunological and biological potencies of the different molecular species of gonadotrophins. Hum. Reprod. 3, 491501. Ulloa-Aguirre, A., Cravioto, A., Damiàn-Matsumura, P. Jimenez, M, Zambrano, E and Diaz-Sanchez, V. (1992) Biological characterization of the naturally occurring analogues of intrapituitary human follicle stimulating hormone. Hum. Reprod. 7, 23-30. Ulloa-Aguirre A, Midgley AR Jr, Beitins IZ, and Pad- manabhan V. (1995). Follicle-stimulating isohormones: characterization and physiological relevance. Endocr Rev.16(6), 765-787. Ulloa-Aguirre A, Maldonado A, Damián-Matsumura P, and Timossi C (2001). Endocrine regulation of gonadotropin glycosylation. Arch Med Res. 32(6), 520-532. Ulloa-Aguirre A, Timossi C, Barrios-de-Tomasi J, Maldonado A, and Nayudu P. (2003). Impact of carbohydrate heterogeneity in function of follicle-stimulating hormone: studies derived from in vitro and in vivo models. Biol Reprod. 69(2), 379-389. Van Lenten L, and Ashwell G. (1972) The binding of desialylated glycoproteins by plasma membranes of rat liver. Develop-ment of a quantitative inhibition assay. J Biol Chem. 247(14), 4633-40. Wide, L. and Albertsson-Wikland, K. (1990) Change in elec-trophoretic mobility of human follicle-stimulating hormone in serum after administration of gonadotropin-releasing hormone. J. Clin. Endocrinol. Metab. 70, 271-276. Wide, L. and Bakos, O. (1993). More basic forms of both human follicle-stimulating hormone and luteinizing hormone in serum at midcycle compared with the follicular or luteal phase. J. Clin. Endocrinol. Metab., 76, 885-889. Wide L, Naessén T, Sundstrom-Poromaa I, Eriksson K. (2007) Sulfonation and sialylation of gonadotropins in women during the menstrual cycle, after menopause, and with polycystic ovarian syndrome and in men. J Clin Endocrinol Metab.;92(11), 4410-4417. Zambrano E, Zarinán T, Olivares A, Barrios-de-Tomasi J, and Ulloa-Aguirre A. (1999). Receptor binding activity and in vitro biological activity of the human FSH charge isoforms as disclosed by heterologous and homologous assay systems: implications for the structure-function relationship of the FSH variants. Endocrine. 10(2), 113121. Zhang X, Lok SH, and Kon OL (1998) Stable expression of human alpha-2,6-sialyltransferase in Chinese hamster ovary cells: functional consequences for human erythropoietin expression and bio-activity. Biochim Biophys Acta. 1425(3), 441-452. SEQ ID NO: 1 Hormônio folículo estimulante alfa polipeptídeo Número de acesso AH007338[00197] The COS protocol proceeds in the usual way regardless of the administration of the initial dose of EF 999049 according to AMH level in line reading with the following table. Thus a patient with an AMH level of 5-14.8 pmol/L would be administered 180 IU FSH in the form of approximately 8-11 μg FE 999049, a human-derived recombinant FSH product manufactured according to the method of Example 6. A patient with an AMH level of 30-44.9 pmol/L would be administered 120 IU FSH in the form of approximately 4-7 μg FE 999049, a human-derived recombinant FSH product manufactured according to the method of Example 6. If the AMH level is not available, the recombinant patient would be administered 120-180 IU FSH in the form of approximately 6-11 μg FE 999049, a human-derived recombinant FSH product manufactured according to the method from Example 6.

References Andersen CY, Westergaard LG, and van Wely M. (2004). FSH isoform composition of commercial gonadotrophin preparations: a neglected aspect? Play Biomed Online. 9(2), 231-236. Arey BJ, Stevis PE, Deecher DC, Shen ES, Frail DE, Negro-Vilar A, and Lopez FJ. (1997) Induction of promiscuous G protein coupling of the follicle-stimulating hormone (FSH) receptor: a novel mechanism for transducing pleiotropic actions of FSH isoforms. Endocrinol Mol. 11(5), 517-526. Baenziger JU and Green ED. (1988). Pituitary glycoprotein hormone oligosaccharides: structure, synthesis and function of the asparagine-linked oligosaccharides on lutropin, follitropin and thyrotropin. Biochim Biophys Acta. 947(2), 287-306. Bassett RM, and Driebergen R. (2005). Continued im-provements in the quality and consistency of follitropin alfa, recombinant human FSH. Play Biomed Online. 10(2), 169-177. Damián-Matsumura P, Zaga V, Maldonado A, Sánchez-Hernández C, Timossi C, and Ulloa-Aguirre A. (1999). Oestrogens regulate pituitary alpha2,3-sialyltransferase messenger ribonucleic acid levels in the female rat. J Mol Endocrinol. 23(2), 153-165. D'Antonio M., Borrelli F., Datola A., Bucci R., Mascia M., Polletta P., Piscitelli D., and Papoian R. (1999) Biological characterization of recombinant human follicle stimulating hormone isoforms. Human Reproduction 14, 1160-1167 Dalpathado DS, Irungu J, Go EP, Butnev VY, Norton K, Bousfield GR, and Desaire H. (2006). Comparative follicle glycomics of the glycoprotein stimulating hormone: glycopeptide analysis of iso-lates from two mammalian species. Biochemistry. 45(28), 8665-8673. Dias JA, Van Roey P. (2001). Structural biology of human follitropin and its receptor. Arch Med Res. 32(6), 510-519 Fiddes, JC and Goodman, HM (1979) Isolation, cloning and sequence analysis of the cDNA for the alpha-subunit of human chorionic gonadotropin. Nature, 281, 351-356. Flack, MR, Bennett, AP, Froehlich, J. Anasti, JN and Nisula, B. (1994). Increased biological activity due to basic isoforms in recombinant human follicle-stimulating hormone produced in a human cell line. J. Clin. Endocrinol. Metab., 79, 756-760 Fox KM, Dias JA, and Van Roey P. (2001). Threedimensional structure of human follicle-stimulating hormone. Endocrinol Mol. 15(3), 378-89 Grabenhorst E, Hoffmann A, Nimtz M, Zettleissl G, and Conradt HS. (1995). Construction of stable BHK-21 cells coexpressing human secretory glycoproteins and human Gal(beta 1-4)GlcNAc-R alpha 2,6-sialyltransferase alpha 2,6-linked NeuAc is preferentially attached to the Gal(beta 1-4)GlcNAc( beta 1-2)Man(alpha 1-3)-branch of frontal oligosaccharides from secreted recombinant beta-trace protein. Eur J Biochem. 232(3), 718-25. Green ED and Baenziger JU. (1988). Asparagine-linked oli-gosaccharides on lutropin, follitropin, and thyrotropin. II. Distributions of sulfated and sialylated oligosaccharides on bovine, ovine, and human pituitary glycoprotein hormones. J Biol Chem. 263(1), 36-44. Grundmann,U., Nerlich,C., Rein,T. and Zettlmeissl, G. (1990). Complete cDNA sequence encoding human beta-galactoside alpha-2,6-sialyltransferase. G Nucleic Acids Res. 18 (3), 667 Howles, CM (1996). Genetic engineering of human FSH (Gonal-F). Hum Play Update, 2172-191. Kagawa Y, Takasaki S, Utsumi J, Hosoi K, Shimizu H, Kochibe N, and Kobata A. (1988). Comparative study of the asparagine-linked sugar chains of natural human interferon-beta 1 and recombinant human interferon-beta 1 produced by three different mammalian cells. J Biol Chem. 263(33), 17508-17515. Keene, JL, Matzuk, MM, Otani, T., Fauser, B,C,J,M., Galway, AB, Hsueh, AJW and Boime, I. (1989). Expression of Biologically active Human Follitropin in Chinese Hamster Ovary Cells. The Journal of Biological Chemistry, 264(9), 4769-4775. Kitagawa, H. and Paulson, JC (1994) Cloning of a novel alpha 2,3-sialyltransferase that sialylates glycoprotein and glycolipid car-bohydrate groups. J. Biol. Chem. 269(2), 1394-1401. Lee EU, Roth J, and Paulson JC (1989) Alteration of terminal glycosylation sequences on N-linked oligosaccharides of Chinese hamster ovary cells by expression of beta-galactoside alpha 2,6-sialyltransferase. J Biol Chem. 264(23), 13848-13855. de Leeuw, R., Mulders, J., Voortman, G. Rombout, F. Damm, J. and Kloosterboer, L. (1996) Structure-function relationship of recombinant follicle stimulating hormone (Puregon). Mol. Hmm. Reprod., 2, 361-369. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. (1951) Protein measurement with the Folin phenol reagent. J Biol Chem. 193(1), 265-75. Lowry, PJ, McLean, C, Jones RL and Satgunasingam N. (1976) Purification of anterior pituitary and hypothalamic hormones Clin Pathol Suppl (Assoc Clin Pathol). 7, 16-21. Olivennes F, Howles CM, Borini A, Germond M, Trew G, Wikland M, Zegers-Hochschild F, Saunders H (2009) Individualizing FSH dose for assisted reproduction using a novel algorithm: the CONSORT study. Play Biomed Online. 2009 Feb;18(2): 195-204. Pierce JG, and Parsons TF (1981) Glycoprotein hormones: structure and function Annu Rev Biochem. 50, 465-495. Pricer WE Jr, and Ashwell G. (1971). The binding of desialylated glycoproteins by plasma membranes of rat liver. J Biol Chem. 246(15), 4825-33. Rathnam P, and Saxena BB. (1975). Primary amino acid sequence of follicle-stimulating hormone from human pituitary glands. I. alpha subunit. J Biol Chem.;250(17):6735-6746. Regoeczi E, Debanne MT, Hatton MC, and Koj A. (1978) Elimination of asialofetuin and asialoorosomucoid by the intact rat. Quantitative aspects of the hepatic clearance mechanism. Biochim Biophys Acta. 541(3), 372-84. Royle L, Radcliffe CM, Dwek RA and Rudd PM (2006) Methods in Molecular Biology, ed I Brockhausen-Schutzbach (Humana Press), 347: Glycobiology protocols, 125-144. Ryan RJ, Keutmann HT, Charlesworth MC, McCormick DJ, Milius RP, Calvo FO and Vutyavanich T. (1987). Structure-function re-lationships of gonadotropins. Recent Prog Horm Res.;43,:383-429. Saxena BB and Rathnam P. (1976) Amino acid sequence of the beta subunit of follicle-stimulating hormone from human pituitary glands. J Biol Chem. 251(4), 993-1005 Steelman SL, and Pohley FM. (1953) Assay of the follicle stimulating hormone based on the augmentation with human chorionic gonadotropin. Endocrinology. 53(6), 604-616. Steer CJ, and Ashwell G. (1980) Studies on a mammalian hepatic binding protein specific for asialoglycoproteins. Evidence for receptor recycling in isolated rat hepatocytes. J Biol Chem. 255(7), 3008-13. Svensson EC, Soreghan B, and Paulson JC. (1990) Organization of the beta-galactoside alpha 2,6-sialyltransferase gene. Evidence for the transcriptional regulation of terminal glycosylation. J Biol Chem. 265(34):20863-20868. Takeuchi M, Takasaki S, Miyazaki H, Kato T, Hoshi S, Kochibe N, and Kobata A (1988). Comparative study of the asparagine-linked sugar chains of human erythropoietins purified from urine and the culture medium of recombinant Chinese hamster ovary cells. J Biol Chem. 263(8), 3657-3663. Timossi CM, Barrios de Tomasi J, Zambrano E, González R, Ulloa-Aguirre A. (1998). A naturally occurring basically charged human follicle-stimulating hormone (FSH) variant inhibits FSH-induced androgen aromatization and tissue-type plasminogen activator enzyme activity in vitro. Neuroendocrinology. 67(3), 153-163. Timossi CM, Barrios-de-Tomasi J, González-Suárez R, Arranz MC, Padmanabhan V, Conn PM, and Ulloa-Aguirre A. (2000). Dif-ferential effects of the charge variants of human follicle-stimulating hormone. J Endocrinol. 165(2), 193-205. Ulloa-Aguirre, A., Espinoza, R., Damian-Matsumura, P. and Chappel, SC (1988) Immunological and biological potencies of the different molecular species of gonadotrophins. Hmm. play 3, 491501. Ulloa-Aguirre A., Cravioto A., Damiàn-Matsumura, P. Jimenez, M, Zambrano, E and Diaz-Sanchez, V. (1992) Biological characterization of the naturally occurring analogues of intrapituitary human follicle stimulating hormone. Hmm. play 7, 23-30. Ulloa-Aguirre A, Midgley AR Jr, Beitins IZ, and Padmanabhan V. (1995). Follicle-stimulating isohormones: characterization and physiological relevance. Endocr Rev.16(6), 765-787. Ulloa-Aguirre A, Maldonado A, Damián-Matsumura P, and Timossi C (2001). Endocrine regulation of gonadotropin glycosylation. Arch Med Res. 32(6), 520-532. Ulloa-Aguirre A, Timossi C, Barrios-de-Tomasi J, Maldonado A, and Nayudu P. (2003). Impact of carbohydrate heterogeneity in function of follicle-stimulating hormone: studies derived from in vitro and in vivo models. Biol Reproduc. 69(2), 379-389. Van Lenten L, and Ashwell G. (1972) The binding of desialylated glycoproteins by plasma membranes of rat liver. Development of a quantitative inhibition assay. J Biol Chem. 247(14), 4633-40. Wide, L. and Albertsson-Wikland, K. (1990) Change in elect-trophoretic mobility of human follicle-stimulating hormone in serum after administration of gonadotropin-releasing hormone. J. Clin. Endocrinol. Metab. 70, 271-276. Wide, L. and Bakos, O. (1993). More basic forms of both human follicle-stimulating hormone and luteinizing hormone in serum at midcycle compared with the follicular or luteal phase. J. Clin. Endocrinol. Metab., 76, 885-889. Wide L, Naessén T, Sundstrom-Poromaa I, Eriksson K. (2007) Sulfonation and sialylation of gonadotropins in women during the menstrual cycle, after menopause, and with polycystic ovarian syndrome and in men. J Clin Endocrinol Metab.;92(11), 4410-4417. Zambrano E, Zarinán T, Olivares A, Barrios-de-Tomasi J, and Ulloa-Aguirre A. (1999). Receptor binding activity and in vitro biological activity of the human FSH charge isoforms as disclosed by heterologous and homologous assay systems: implications for the structure-function relationship of the FSH variants. endocrine. 10(2), 113121. Zhang X, Lok SH, and Kon OL (1998) Stable expression of human alpha-2,6-sialyltransferase in Chinese hamster ovary cells: functional consequences for human erythropoietin expression and bio-activity. Biochim Biophys Acta. 1425(3), 441-452. SEQ ID NO: 1 Alpha Polypeptide Follicle Stimulating Hormone Accession Number AH007338

Claims (5)

1. Composition, characterized in that it comprises recombinant follicle stimulating hormone (rFSH) including α2,3- and α2,6 sialylation, wherein rFSH includes mono-, di-, tri- and tetra-sialylated glycan structures, in that 15 to 24% of sialylated glycan structures are tetrasialylated glycan structures. 2. Composition, according to claim 1, characterized in that the rFSH glycans also comprise 20% or more of N-Acetylgalactosamine (GalNAc). 3. Composition, characterized in that it comprises 12 μg, 15 μg, 18 μg or 24 μg of recombinant FSH, as defined in claim 1 or 2. 4. Composition according to claim 3, characterized in that it further comprises a GnRH antagonist. 5. Use of recombinant follicle stimulating hormone (rFSH), as defined in claim 1 or 2, characterized in that it is for the manufacture of a drug for the treatment of infertility.

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